Replikin peptides and antibodies therefore

ABSTRACT

The present invention provides a new class of peptides related to rapid replication and their use in diagnosing, preventing and treating disease.

CROSS REFERENCE TO OTHER APPLICATIONS

This application is a Continuation-In-Part of application No. U.S. Ser.No. 10/105,232, filed Mar. 26, 2002, which is a Continuation-In-Part ofU.S. Ser. No. 09/984,057, filed Oct. 26, 2001, which claims priorityfrom Provisional Applications 60/303,396, filed Jul. 9, 2001 and60/278,761 filed Mar. 27, 2001, the subject matter of which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the identification and use of Replikins, anewly discovered class of peptides that share structuralcharacteristics. In particular, this invention relates to Replikinswhich have been found in viruses, bacteria, fungus, cancer associatedproteins, plants and unicellular parasites and their use as targets inthe development of methods of treating or preventing diseases. Further,this invention relates to the use of Replikins in the detection of thesediseases. Also this invention relates to the use of Replikins tostimulate growth of plants used for food.

INTRODUCTION AND BACKGROUND OF THE INVENTION

Rapid replication is characteristic of virulence in certain bacteria,viruses and malignancies, but no chemistry common to rapid replicationin different organisms has been described previously. This patentapplication discloses a new class of protein structures related to rapidreplication. A new family of conserved small proteins related to rapidreplication, named Replikins, which are used to predict and controlrapid replication in multiple organisms and diseases and to induce rapidreplication in plant and animal life.

We constructed an algorithm search for Replikins. In applying thealgorithm invented herein not only was the function of the epitoperevealed—rapid replication, but an entire family of homologues whosefunction is related to rapid replication was discovered, which we namedReplikins.

The algorithm is based on the following: 1) Evidence that the immunesystem looks to parts rather than a whole protein in recognition.Protein chains are first hydrolyzed by the immune system into smallerpieces, frequently six (6) to ten (10) amino acids long, as part of theimmune systems' process of recognition of foreign structures againstwhich it may mount an immune defense. By way of example, the immunesystem recognizes the presence of disease by chopping up proteins of thedisease agent into smaller peptide sequences and reading them. Thisprinciple is used as a basis for the algorithm with which to search forhomologues of the malignin cancer epitope, once the structure of theepitope was known; 2) The specific structure of the malignin epitope, inwhich two of the three lysines (K's) are eight residues apart is inaccordance with the apparent ‘rules’ used by the immune system forrecognition referred to above (6-10 amino acids long); 3) The fact thatthe malignin cancer epitope was shown to be a very strong antigen, thatis—a generator of a strong immune response; that there are three lysines(K's) in the 10-mer peptide glioma Replikin and that K's are known tobind frequently to DNA and RNA as potential anchors for the entry ofviruses; and 4) One histidine (H) is included in the sequence of themalignin epitope, between the two K's which are eight (8) residuesapart, suggesting a connection to the metals of redox systems which arerequired to provide the energy for replication.

Engineered enzymes and catalytic antibodies, possessing tailored bindingpockets with appropriately positioned functional groups have beensuccessful in catalyzing a number of chemical transformations, sometimeswith impressive efficiencies. Just as two or more separate proteins withspecific and quite different functions are now often recognized to besynthesized together by organisms, and then separately cleaved to ‘goabout their separate functions’, so the Replikin structure is a uniqueprotein with a unique function that appears to be recognized separatelyby the immune system and may be now rationally engineered—e.g.synthesized to produce a functional unit.

From a proteomic point of view, this template based on the newlydetermined glioma peptide sequence has led to the discovery of a wideclass of proteins with related conserved structures and a particularfunction, in this case replication. Examples of the increase in Replikinconcentration with virulence of a disease appear in diseases including,influenza, HIV, cancer and tomato leaf curl virus. This class ofstructures is related to the phenomenon of rapid replication inorganisms as diverse as yeast, algae, plants, the gemini curl leaftomato virus, HIV and cancer.

In addition to detecting the presence of Replikins in rapidlyreplicating organisms, we found that 1) Replikin concentration (numberof Replikins per 100 amino acids) and 2) Replikin compositions inspecific functional states dependant on rapid replication, provide thebasis for the finding that Replikins are related quantitatively as wellas qualitatively to the rate of replication of the organism in whichthey reside. Examples of these functional proofs include therelationship found between rapid replication and virulence inglioblastoma cells, between Replikins in influenza virus and theprediction of influenza pandemics and epidemics, and the relationshipbetween Replikin concentration and rapid replication in HIV.

The first functional basis for Replikins' role in rapid replication wasfound in the properties of the glioma Replikin, a 10 KD peptide calledMalignin in brain glioblastoma multiforme (glioma)—a 250 KD cellprotein. Antimalignin antibody increased in concentration in serum(AMAS), measured by an early stage diagnostic test for cancer now usedfor most or all cell types. Malignin was so named because in tissueculture the expression of this peptide and its concentration permilligram membrane protein extractable increased with increased rate ofcell division per unit time. Not only is there an increase in the amountof malignin in proportion to the cell number increase but the amount ofmalignin is enriched, that is—increased ten fold whereas the cell numberincreased only five fold.

The structure of malignin protein was determined through hydrolysis andmass spectrometry which revealed what proved to be a novel 16 merpeptide sequence. We searched for the 16 mer peptide sequence which wehave named a Glioma Replikin protein in databases for the healthy humangenome and found that it was not present in these databases.

As such, the fixed requirement algorithm was used to search in otherorganisms for the Glioma Replikin protein or homologues thereof. Over4,000 protein sequences in the “Pub Med” database were searched andhomologues were found in viruses and plant forms specifically associatedwith rapid replication. Homologues of such Replikin proteins occurredfrequently in proteins called ‘replicating proteins’ by theirinvestigators.

Homologues of the Replikin sequence were found in all tumor viruses(that is viruses that cause cancer), and in ‘replicating proteins’ ofalgae, plants, fungi, viruses and bacteria.

That malignin is enriched ten-fold compared to the five-fold increase incell number and membrane protein concentration in rapid replication ofglioma cells suggests an integral relationship of the Replikins toreplication. When the glioma replikin was synthesized in vitro andadministered as a synthetic vaccine to rabbits, abundant antimaligninantibody was produced—establishing rigourously the antigenic basis ofthe antimalignin antibody in serum (AMAS) test, and providing the firstpotential synthetic cancer vaccine and the prototype for Replikinvaccines in other organisms.

The demonstration of the relationship of the Replikins to replicationand the natural immune response to cancer Replikins (overriding celltype) based upon the shared specificity of cancer Replikins, permitspassive augmentation of immunity with antimalignin antibody and activeaugmentation with synthetic Replikin vaccines.

A study of 8,090 serum specimens from cancer patients and controls hasdemonstrated that the concentration of antimalignin antibody increaseswith age in healthy individuals, as the incidence of cancer in thepopulation increases, and increases further two to three-fold in earlymalignancy, regardless of cell type. In vitro this antibody is cytotoxicto cancer cells at picograms (femtomoles) per cancer cell, and in vivothe concentration of antimalignin antibody relates quantitatively to thesurvival of cancer patients. As shown in glioma cells, the stage incancer at which cells only have been transformed to the immortalmalignant state but remain quiescent or dormant, now can bedistinguished from the more active life-threatening replicating statewhich is characterized by the increased concentration of Replikins. Inaddition, clues to the viral pathogenesis of cancer may be found in thefact that glioma glycoprotein 10B has a 50% reduction in carbohydrateresidues when compared to the normal 10B. This reduction is associatedwith virus entry in other instances and so may be evidence of theattachment of virus for the delivery of virus Replikins to the 10B ofglial cells as a step in the transformation to the malignant state.

The sharing of immunological specificity by diverse members of theclass, as demonstrated with antimalignin antibody for the glioma andrelated cancer Replikins, suggests that B cells and their productantibodies may recognize Replikins by means of a similar recognition‘language’. With the discovery of the Replikins, this sharedimmunological specificity may explain what was previously difficult tounderstand: why the antimalignin antibody is elevated in all cancers,and is cytotoxic to cancer cells and related to survival of cancerpatients in most or all cell types. Thus antimalignin antibody isproduced against cancer Replikins, which share immunological specificityand which are related to the phenomenon of rapid replication, not tocell type.

A second functional basis for the Replikins' role in rapid replicationis the study of data from the past 100 years on influenza virushemagglutinin protein sequences and epidemiology of influenza epidemicsand pandemics. To date, only serological hemagglutinin and antibodyclassification, but no strain-specific conserved peptide sequences havepreviously been described in influenza, and no changes in concentrationand composition of any strain-specific peptide sequences have beendescribed previously which correlate with epidemiologically documentedepidemics or rapid replication.

A four to ten-fold increase in the concentration of strain-specificinfluenza Replikins in one of each of the four major strains, influenzaB, (A)H1N1, (A)H2N2 and (A)H3N2 was found, and that such increase ofReplikin concentration was related to influenza epidemics causedspecifically by each strain from 1902 to 2001. These increases inconcentration were then shown to be due to the reappearance of at leastone specific Replikin composition from 1 to up to 64 years after itsdisappearance, plus the emergence of new strain-specific Replikincompositions. Previously, no strain-specific chemical structures wereknown with which to predict which strains would predominate in cominginfluenza seasons, nor to devise annual mixtures of whole-virus strainsfor vaccines. The recent sharp increase in H3N2 Replikin concentration(1997 to 2000), the largest in H3N2's history, and the reappearance ofspecific Replikin compositions which were last seen in the highmortality H3N2 pandemic of 1968 and in the two high mortality epidemicsof 1975 and 1977, but were absent for 20-25 years, together may be awarning of coming epidemics.

Synthetic Replikins are new vaccines. This high degree of conservationof Replikin structures observed whereby the identical structure canpersist for 100 years, or reappear after an absence of from one to 64years reappears indicates that what was previously thought to be changein virulence due to random substitution of amino acids in influenzaproteins is more likely to be change due to an organized process ofconservation of Replikins. In fact, if random substitutions of eachamino acid occurred, the chance against an average length influenzaReplikin sequence being conserved for one year (let alone 100) iscalculated to be in the order of 2 to the 27^(th) power to 1.

The significant conservation of Replikins is not unique to influenzavirus is also present in foot and mouth disease virus type O and in HIV,as well as in wheat.

A third functional basis for Replikins' role in rapid replication is theincrease in Replikin concentration shown to be related to rapidreplication in HIV. The Replikin concentration in the slow-growinglow-titre strain of HIV (NS1, “Bru”), prevalent in early stageinfection, was found to be one-sixth of the Replikin concentration inthe rapidly-growing high-titre strain of HIV (SI, “Lai”), prevalent inlate stage HIV infection.

Other examples are given of the relation of Replikins to rapidreplication. For example, in tomato curl leaf gemini virus, whichdevastates tomato crops, the first 161 amino acids of the ‘replicatingprotein’, which have been shown to bind to DNA, contain five Replikins.

In malaria, legendary for rapid replication, trypanosomes are releasedfrom the liver in tens of thousands from one trypanosome. Multiple,novel, almost ‘flamboyant’ Replikin structures with concentrations of upto 36 overlapping Replikins per 100 amino acids are found therein.

The increase in Replikin concentration in influenza epidemics isfunctionally comparable to the glioma Replikin's increase inconcentration during rapid replication of malignant glioma cells andcomparable to rapid replication in HIV and in a diverse range of otherorganisms. Replikins thus are associated with and appear to be part ofthe structural bases of rapid replication in different organisms.

Replikin concentration and composition therefore provide new methods todetect and to control the process of replication, which is central tothe survival and dominance of each biological population. The discoveryof these new proteins related to rapid replication provides newopportunities 1) for detection of pathogens by qualitative andquantitative determinations of Replikins, 2) for the control of a broadrange of diseases in which rapid replication is a key factor bytargeting native Replikins and by using synthetic Replikins as vaccines,and 3) for the use of Replikins to foster growth of algal and plantfoods.

There is a significant number of diseases and pathogens which haveproved difficult to detect and treat and for which there is no effectivevaccine. Thus, for each disorder there is a need for developing a targetthat will provide effective methods of detecting, treating or preventingthese diseases and pathogens.

SUMMARY OF THE INVENTION

The present invention provides a method for identifying nucleotide oramino acid sequences that include a Replikin sequence. The method isreferred to herein as a 3-point-recognition method. By use of the“3-point recognition” method, namely, peptides comprising from 7 toabout 50 amino acids including:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues (Replikin)—constituting a new        class of peptides was revealed in algae, yeast, fungi, amoebae,        bacteria, plant and virus proteins having replication,        transformation, or redox functions.

In one aspect of the invention there are provided isolated orsynthesized peptides containing a Replikin sequence. The peptidescomprise from 7 to about 50 amino acids including:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residues;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

The present invention also provides methods for detecting the presenceof a contaminating organism in a body sample or environmental samplecomprising:

-   -   (1) isolating nucleic acids from the body sample or        environmental sample;    -   (2) screening the nucleic acids for the presence of a Replikin        structure; and    -   (3) correlating the presence of a Replikin structure with the        presence of the contaminating organism.

In another aspect of the invention there is provided a process forstimulating the immune system of a subject to produce antibodies thatbind specifically to a Replikin sequence, said process comprisingadministering to the subject an effective amount of a dosage of acomposition comprising at least one Replikin peptide. One embodimentcomprises at least one peptide that is present in an emerging strain ofthe organism if such new strain emerges.

The present invention also provides antibodies that bind specifically toa Replikin, as defined herein, as well as antibody cocktails containinga plurality of antibodies that specifically bind to Replikins. In oneembodiment of the invention, there are provided compositions comprisingan antibody or antibodies that specifically bind to a Replikin and apharmaceutically acceptable carrier.

In one aspect of the invention there are provided isolated, or separatedfrom other proteins, recombinant, or synthesized peptides or othermethods containing a viral Replikin sequence. The viral Replikinpeptides comprise from 7 to about 50 amino acids including:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues. (viral Replikin).

The present invention also provides methods for detecting the presenceof a contaminating virus in a body sample or environmental samplecomprising:

-   -   (1) isolating nucleic acids from the body sample or        environmental sample;    -   (2) screening the nucleic acids for the presence of a viral        Replikin structure; and    -   (3) correlating the presence of viral Replikin structures, their        concentration and composition, with the presence of the        contaminating virus.

In another aspect of the invention there is provided a process forstimulating the immune system of a subject to produce antibodies thatbind specifically to a viral Replikin sequence, said process comprisingadministering to the subject an effective amount of a dosage of acomposition comprising at least one Replikin peptide. One embodimentcomprises at least one peptide that is present in an emerging strain ofthe virus if such new strain emerges.

The present invention also provides antibodies that bind specifically toa viral Replikin, as defined herein, as well as antibody cocktailscontaining a plurality of antibodies that specifically bind to viralReplikins. In one embodiment of the invention, there are providedcompositions comprising an antibody or antibodies that specifically bindto a viral Replikin and a pharmaceutically acceptable carrier.

The present invention also provides therapeutic compositions comprisingone or more of isolated virus peptides having from 7 to about 50 aminoacids comprising:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues, and a pharmaceutically        acceptable carrier.

In another aspect of the invention there is provided an antisensenucleic acid molecule complementary to a virus Replikin mRNA sequence,said Replikin mRNA sequence denoting from 7 to about 50 amino acidscomprising:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

In yet another aspect of the invention there is provided a method ofsimulating the immune system of a subject to produce antibodies toviruses, said method comprising: administering an effective amount of atleast one virus Replikin peptide having from 7 to about 50 amino acidscomprising:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

In another aspect, there is provided a method of selecting a viruspeptide for inclusion in a preventive or therapeutic virus vaccinecomprising:

-   -   (1) obtaining at least one isolate of each strain of a plurality        of strains of said virus;    -   (2) analyzing the amino acid sequence of the at least one        isolate of each strain of the plurality of strains of the virus        for the presence and concentration of Replikin sequences;    -   (3) comparing the concentration of Replikin sequences in the        amino acid sequence of the at least one isolate of each strain        of the plurality of strains of the virus to the concentration of        Replikin sequences observed in the amino acid sequence of each        of the strains at least one earlier time period to provide the        concentration of Replikins for at least two time periods, said        at least one earlier time period being within about six months        to about three years prior to step (1);    -   (4) identifying the strain of the virus having the highest        increase in concentration of Replikin sequences during the at        least two time periods; and    -   (5) selecting at least one Replikin sequence present in the        strain of the virus peptide identified in step (4) as a peptide        for inclusion in the virus vaccine.

The present invention also provides a method of making a preventive ortherapeutic virus vaccine comprising:

-   -   (1) identifying a strain of a virus as an emerging strain,    -   (2) selecting at least one Replikin sequence present in the        emerging strain as a peptide template for the virus vaccine        manufacture,    -   (3) synthesizing peptides having the amino acid sequence of the        at least one Replikin sequence selected in step (2), and    -   (4) combining a therapeutically effective amount of the peptides        of step (3) with a pharmaceutically acceptable carrier and/or        adjuvant.

In another aspect, the invention is directed to a method of identifyingan emerging strain of a virus for diagnostic, preventive or therapeuticpurposes comprising:

-   -   (1) obtaining at least one isolate of each strain of a plurality        of strains of the virus;    -   (2) analyzing the amino acid sequence of the at least one        isolate of each strain of the plurality of strains of the virus        for the presence and concentration of Replikin sequences;    -   (3) comparing the concentration of Replikin sequences in the        amino acid sequence of the at least one isolate of each strain        of the plurality of strains of the virus to the concentration of        Replikin sequences observed in the amino acid sequence of each        of the strains at least one earlier time period to provide the        concentration of Replikins for at least two time periods, said        at least one earlier time period being within about six months        to about three years prior to step (1); and    -   (4) identifying the strain of the virus having the highest        increase in concentration of Replikin sequences during the at        least two time periods.

In yet another aspect of the invention, there is provided a preventiveor therapeutic virus vaccine comprising at least one isolated Replikinpresent in a protein of an emerging strain of the virus and apharmaceutically acceptable carrier and/or adjuvant.

Also provided by the present invention is a method of preventing ortreating a virus infection comprising administering to a patient in needthereof a preventive or therapeutic virus vaccine comprising at leastone isolated Replikin present in a protein of an emerging strain of thevirus and a pharmaceutically acceptable carrier and/or adjuvant.

Influenza

Influenza is an acute respiratory illness of global importance. Despiteinternational attempts to control influenza virus outbreaks throughvaccination, influenza infections remain an important cause of morbidityand mortality. Worldwide influenza epidemics and pandemics have occurredat irregular and previously unpredictable intervals throughout historyand it is expected that they will continue to occur in the future. Theimpact of both pandemic and epidemic influenza is substantial in termsof morbidity, mortality and economic cost.

Influenza vaccines remain the most effective defense against influenzavirus, but because of the ability of the virus to mutate and theavailability of non-human host reservoirs, it is expected that influenzawill remain an emergent or re-emergent infection. Global influenzasurveillance indicates that influenza viruses may vary within a countryand between countries and continents during an influenza season.Virological surveillance is of importance in monitoring antigenic shiftand drift. Disease surveillance is also important in assessing theimpact of epidemics. Both types of information have provided the basisof the vaccine composition and the correct use of antivirals. However,to date there has been only annual post hoc hematological classificationof the increasing number of emerging influenza virus strains, and nospecific chemical structure of the viruses has been identified as anindicator of approaching influenza epidemics or pandemics. Currently,the only basis for annual classification of influenza virus as active,inactive or prevalent in a given year is the activities of the virushemagglutinin and neuraminidase proteins. No influenza viral chemicalstructure has been identified prior to this application that can be usedfor quantitative warning of epidemics or pandemics or to design moreeffective and safer vaccines.

Because of the annual administration of influenza vaccines and the shortperiod of time when a vaccine can be administered, strategies directedat improving vaccine coverage are of critical importance.

In one aspect of the invention there are provided isolated orsynthesized influenza virus peptides containing a Replikin sequence. Theinfluenza Replikin virus peptides comprise from 7 to about 50 aminoacids including:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues. (Influenza Replikin).

In another aspect of the invention, there is provided a process forstimulating the immune system of a subject to produce antibodies thatbind specifically to an influenza virus Replikin sequence, said processcomprising administering to the subject an effective amount of dosage ofa composition comprising at least one influenza virus Replikin peptide.In a preferred embodiment the composition comprises at least on peptidethat is present in an emerging strain of influenza virus.

The present invention also provides antibodies that bind specifically toan influenza virus Replikin, as defined herein, as well as antibodycocktails containing a plurality of antibodies that specifically bind toinfluenza virus Replikins. In one embodiment of the invention, there areprovided compositions comprising an antibody or antibodies thatspecifically bind to an influenza Replikin and a pharmaceuticallyacceptable carrier.

The present invention also provides therapeutic compositions comprisingone or more of isolated influenza virus peptides having from 7 to about50 amino acids comprising:

-   -   (1) at least one lysine residue located six to ten residues form        a second lysinc residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues, and a pharmaceutical acceptable        carrier.

In another aspect of the invention there is provided an antisensenucleic acid molecule complementary to an influenza virus hemagglutininReplikin mRNA sequence, said Replikin mRNA sequence denoting from 7 toabout 50 amino acids comprising:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

In yet another aspect of the invention there is provided a method ofsimulating the immune system of a subject to produce antibodies toinfluenza virus comprising administering an effective amount of at leastone influenza virus Replikin peptide having from 7 to about 50 aminoacids comprising:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

In another aspect, there is provided a method of selecting an influenzavirus peptide for inclusion in a preventive or therapeutic influenzavirus vaccine comprising:

-   -   (1) obtaining at least one isolate of each strain of a plurality        of strains of influenza virus;    -   (2) analyzing the hemagglutinin amino acid sequence of the at        least one isolate of each strain of the plurality of strains of        influenza virus for the presence and concentration of Replikin        sequences;    -   (3) comparing the concentration of Replikin sequences in the        hemagglutinin amino acid sequence of the at least one isolate of        each strain of the plurality of strains of influenza virus to        the concentration of Replikin sequences observed in the        hemagglutinin amino acid sequence of each of the strains at        least one earlier time period to provide the concentration of        Replikins for at least two time periods, said at least one        earlier time period being within about six months to about three        years prior to step (1);    -   (4) identifying the strain of influenza virus having the highest        increase in concentration of Replikin sequences during the at        least two time periods;    -   (5) selecting at least one Replikin sequence present in the        strain of influenza virus peptide identified in step (4) as a        peptide for inclusion in an influenza virus vaccine.

The present invention also provides a method of making a preventive ortherapeutic influenza virus vaccine comprising:

-   -   (1) identifying a strain of influenza virus as an emerging        strain;    -   (2) selecting at least one Replikin sequence present in the        emerging strain as a peptide template for influenza virus        vaccine manufacture,    -   (3) synthesizing peptides having the amino acid sequence of the        at least one Replikin sequence selected in step (2), and    -   (4) combining a therapeutically effective amount of the peptides        of step (3) with a pharmaceutically acceptable carrier and/or        adjuvant.

In another aspect, the invention is directed to a method of identifyingan emerging strain of influenza virus for diagnostic, preventive ortherapeutic purposes comprising:

-   -   (1) obtaining at least one isolate of each strain of a plurality        of strains of influenza virus;    -   (2) analyzing the hemagglutinin amino acid sequence of the at        least one isolate of each strain of the plurality of strains of        influenza virus for the presence and concentration of Replikin        sequences;    -   (3) comparing the concentration of Replikin sequences in the        hemagglutinin amino acid sequence of the at least one isolate of        each strain of the plurality of strains of influenza virus to        the concentration of Replikin sequences observed in the        hemagglutinin amino acid sequence of each of the strains at        least one earlier time period to provide the concentration of        Replikins for at least two time periods, said at least one        earlier time period being within about six months to about three        years prior to step (1); and    -   (4) identifying the strain of influenza virus having the highest        increase in concentration of Replikin sequences during the at        least two time periods.

In yet another aspect of the invention, there is provided a preventiveor therapeutic influenza virus vaccine comprising at least one isolatedReplikin present in the hemagglutinin protein of an emerging strain ofinfluenza virus and a pharmaceutically acceptable carrier and/oradjuvant.

Also provided by the present invention is a method of preventing ortreating influenza virus infection comprising administering to a patientin need thereof a preventive or therapeutic vaccine comprising at leastone isolated Replikin present in the hemagglutinin protein of anemerging strain of influenza virus and a pharmaceutically acceptablecarrier and/or adjuvant.

Trypanosomes

In one aspect of the invention there are provided isolated orsynthesized trypanosome peptides containing a Replikin sequence. Thetrypanosome Replikin peptides comprise from 7 to about 50 amino acidsincluding:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues. (Trypanosome Replikins).

Malaria

One trypanosome disorder which has proved difficult to treat and forwhich there is no effective vaccine is malaria. Malaria causes muchdeath, and physical and economic hardship in tropical regions. Malariais caused mainly by Plasmodium falciparum, which has proved to beextremely resistant to treatment and to date, a vaccine for malaria hasremained elusive. Thus there is a need for effective malaria vaccinesand methods of treating or preventing the disease. This applicationprovides the basis for such vaccines and methods of treatment andprevention. All of the methods described above for production of andtreatment with Replikin virus vaccines and Replikin influenza virusvaccines are applicable to the production of and treatment with Replikinmalaria vaccines.

In the present invention, there are provided vaccines and methods forpreventing or treating malaria. The malaria vaccines comprise at leastone isolated Plasmodium falciparum Replikin. The present invention alsoprovides methods for treating or preventing malaria comprisingadministering to a patient an effective amount of preventive ortherapeutic vaccine comprising at least one isolated Plasmodiumfalciparum Replikin.

Also provided by the present invention are antibodies, antibodycocktails and compositions that comprise antibodies that specificallybind to a Replikin or Replikins present in a malaria antigen ofPlasmodium falciparum.

Another example of a trypanosome which may be treated under the presentinvention as is the case for malaria, the Replikins of TreponemaPallidum (syphilis), can be used for detection, prevention, treatment ofsyphilis.

Bacteria

In one aspect of the invention there are provided isolated orsynthesized bacterial peptides containing a Replikin sequence (bacterialReplikins). The bacterial peptides comprise from 7 to about 50 aminoacids including:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues. (bacterial Replikins). U.S.        application Ser. No. 10/105,232 filed Mar. 26, 2002 is        incorporated by reference in its entirety, including but not        limited to the bacterial sequence listing and information.

The present invention also provides methods for detecting the presenceof a contaminating bacterial organism in a body sample or environmentalsample comprising:

-   -   (1) isolating nucleic acids from the body sample or        environmental sample;    -   (2) screening the nucleic acids for the presence of a Replikin        structure; and    -   (3) correlating the presence of a Replikin structure with the        presence of the contaminating organism.

In another aspect of the invention there is provided a process forstimulating the immune system of a subject to produce antibodies thatbind specifically to a bacterial Replikin sequence, said processcomprising administering to the subject an effective amount of a dosageof a composition comprising at least one bacterial Replikin peptide. Oneembodiment comprises at least one bacterial peptide that is present inan emerging strain of the bacterial organism if such new strain emerges.

The present invention also provides antibodies that bind specifically toa bacterial Replikin, as defined herein, as well as antibody cocktailscontaining a plurality of antibodies that specifically bind to bacterialReplikins. In one embodiment of the invention, there are providedcompositions comprising an antibody or antibodies that specifically bindto a bacterial Replikin and a pharmaceutically acceptable carrier.

The present invention also provides therapeutic compositions comprisingone or more of isolated bacterial peptides having from 7 to about 50amino acids comprising:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue;    -   (3) at least 6% lysine residues; and    -   (4) a pharmaceutically acceptable carrier.

In another aspect of the invention there is provided an antisensenucleic acid molecule complementary to a bacterial Replikin mRNAsequence, said Replikin mRNA sequence denoting from 7 to about 50 aminoacids comprising:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

In yet another aspect of the invention there is provided a method ofsimulating the immune system of a subject to produce antibodies tobacteria comprising administering an effective amount of at least onebacterial Replikin peptide having from 7 to about 50 amino acidscomprising:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

In another aspect, there is provided a method of selecting a bacterialReplikin peptide for inclusion in a preventive or therapeutic bacterialvaccine comprising:

-   -   (1) obtaining at least one isolate of each strain of a plurality        of strains of the bacteria;    -   (2) analyzing the amino acid sequence of the at least one        isolate of each strain of the plurality of strains of the        bacteria for the presence and concentration of bacterial        Replikin sequences;    -   (3) comparing the concentration of bacterial Replikin sequences        in the amino acid sequence of the at least one isolate of each        strain of the plurality of strains of the bacteria to the        concentration of bacterial Replikin sequences observed in the        amino acid sequence of each of the strains at least one earlier        time period to provide the concentration of bacterial Replikins        for at least two time periods, said at least one earlier time        period being within about six months to about three years prior        to step (1), or earlier in rapidly mutating bacteria;    -   (4) identifying the strain of the bacteria having the highest        increase in concentration of bacterial Replikin sequences during        the at least two time periods; and    -   (5) selecting at least one bacterial Replikin sequence present        in the strain of the bacterial peptide identified in step (4) as        a peptide for inclusion in the bacterial vaccine.

The present invention also provides a method of making a preventive ortherapeutic bacterial vaccine comprising:

-   -   (1) identifying a strain of a bacteria as an emerging strain;    -   (2) selecting at least one bacterial Replikin sequence present        in the emerging strain as a peptide template for the bacterial        vaccine manufacture;    -   (3) synthesizing peptides having the amino acid sequence of the        at least one bacterial Replikin sequence selected in step (2);        and    -   (4) combining a therapeutically effective amount of the peptides        of step (3) with a pharmaceutically acceptable carrier and/or        adjuvant.

In another aspect, the invention is directed to a method of identifyingan emerging strain of bacteria for diagnostic, preventive or therapeuticpurposes comprising:

-   -   (1) obtaining at least one isolate of each strain of a plurality        of strains of the bacteria;    -   (2) analyzing the amino acid sequence of the at least one        isolate of each strain of the plurality of strains of the        bacteria for the presence and concentration of bacterial        Replikin sequences;    -   (3) comparing the concentration of bacterial Replikin sequences        in the amino acid sequence of the at least one isolate of each        strain of the plurality of strains of the bacteria to the        concentration of bacterial Replikin sequences observed in the        amino acid sequence of each of the strains at least one earlier        time period to provide the concentration of bacterial Replikins        for at least two time periods, said at least one earlier time        period being within about six months to about three years prior        to step (1); and    -   (4) identifying the strain of the bacteria having the highest        increase in concentration of bacterial Replikin sequences during        the at least two time periods.

In yet another aspect of the invention, there is provided a preventiveor therapeutic bacterial vaccine comprising at least one isolatedbacterial Replikin present in a protein of an emerging strain of thebacteria and a pharmaceutically acceptable carrier and/or adjuvant.

Two important sub-species of bacteria, classified under mycobacteria,are Mycobacterium leprae (leprosy) whose 30-s ribosomal protein has aC-terminal Replikin and Mycobacterium tuberculosis (tuberculosis) whoseATPase has three Replikins:

Replikin in 30s ribosomal protein s6 of Mycobacterium leprae (leprosy)is: kvmrtdkh (SEQ ID NO. 699) Replikins in the ATPase of Mycobacteriumtuberculosis are: hprpkvaaalkdsyrlk (SEQ ID NO. 700) hprpkvaaalk (SEQ IDNO. 701) ksaqkwpdkflagaaqvah (SEQ ID NO. 702) Replikins in theB-D-galactosidase of E. coli: hawqhqgktlfisrk (SEQ ID NO. 703)hqgktlfisrk (SEQ ID NO. 704) Replikins in Agrobacterium tumefaciens:hsdqqlavmiaakrlddyk (SEQ ID NO. 705) hlldhpasvgqldlramlaveevkidnpvymek(SEQ ID NO. 706) hpasvgqldlramlaveevkidnpvymek (SEQ ID NO. 707)kcvmakncnikcpaglttnqeafngdpralaqylmniah (SEQ ID NO. 708)kncnikcpaglttnqeafngdpralaqylmniah (SEQ ID NO. 709)hhdtysiedlaqlihdakaarvrvivk (SEQ ID NO. 710) hdtysiedlaqlihdakaarvrvivk(SEQ ID NO. 711) hdakaarvrvivk (SEQ ID NO. 712)kigqgakpgeggqlpspkvtvciaaarggtpgvelvsppphh (SEQ ID NO. 713)kigqgakpgeggqlpspkvtveiaaarggtpgvelvsppph (SEQ ID NO. 714)kaseitktlasgamshgalvaaaheavahgtnmvggmsnsgeggeh (SEQ ID NO. 715)kaseitktlasgamshgalvaaaheavah (SEQ ID NO. 716) kaseitktlasgamshgalvaaah(SEQ ID NO. 717) kaseitktlasgamsh (SEQ ID NO. 718)kryfpnvktpvggvtfaviaqavadwh (SEQ ID NO. 719)hhiaaglgfgasavyplgvqfraeekfgadadkafkrfakaackslmk (SEQ ID NO. 720)hhiaaglgfgasavyplgvqfraeekfgadadkafkrfakaackslmk (SEQ ID NO. 721)hhiaaglgfgasavyplgvqfraeekfgadadkafkrfakaaek (SEQ ID NO. 722)hhiaaglgfgasavyplgvqfraeekfgadadkafkrfak (SEQ ID NO. 723)hhiaaglgfgasavyplgvqfraeekfgadadk (SEQ ID NO. 724)hiaaglgfgasavyplgvqfraeekfgadadkafktfakaaekslmk (SEQ ID NO. 725)hiaaglgfgasavyplgvqfraeekfgadadkafkrfakaaek (SEQ ID NO. 726)hiaaglgfgasavyplgvqfraeekfgadadkafkrfak (SEQ ID NO. 727)hiaaglgfgasavyplgvqfraeekfgadadk (SEQ ID NO. 728)kfglydaafeksscgvgfitrkdgvqth (SEQ ID NO. 729)

Also provided by the present invention is a method of preventing ortreating a bacterial infection comprising administering to a patient inneed thereof a preventive or therapeutic vaccine comprising at least oneisolated bacterial Replikin present in a protein of an emerging strainof the bacteria and a pharmaceutically acceptable carrier and/oradjuvant.

Fungus

In one aspect of the invention there are provided isolated orsynthesized fungal peptides containing a Replikin sequence. The fungalReplikin peptides comprise from 7 to about 50 amino acids including:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues (fungal Replikins).

All of the methods described above for production of and treatment withbacterial Replikin vaccines are applicable to the production of andtreatment with fungal Replikin vaccines.

In another aspect of the invention there is provided a process forstimulating the immune system of a subject to produce antibodies thatbind specifically to a fungal Replikin sequence, said process comprisingadministering to the subject an effective amount of a dosage of acomposition comprising at least one fungal Replikin peptide.

The present invention also provides antibodies that bind specifically toa fungal Replikin, as defined herein, as well as antibody cocktailscontaining a plurality of antibodies that specifically bind to viralReplikins. In one embodiment of the invention, there are providedcompositions comprising an antibody or antibodies that specifically bindto a fungal Replikin and a pharmaceutically acceptable carrier.

The present invention also provides therapeutic compositions comprisingone or more of isolated fungal peptides having from 7 to about 50 aminoacids comprising:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue;    -   (3) at least 6% lysine residues; and    -   (4) a pharmaceutically acceptable carrier.

In another aspect of the invention there is provided an antisensenucleic acid molecule complementary to an fungal Replikin mRNA sequence,said Replikin mRNA sequence having from 7 to about 50 amino acidscomprising:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

In another aspect of the invention there is provided a process forstimulating the immune system of a subject to produce antibodies thatbind specifically to a fungal Replikin sequence, said process comprisingadministering to the subject an effective amount of a dosage of acomposition comprising at least one Replikin peptide.

Increasing Replication

In yet another aspect of the invention there is provided a method forincreasing the replication rate of an organism comprising transforming agene encoding an enzyme or other protein having a replication functionin the organism with at least one Replikin structure.

DEFINITIONS

As used herein, the term “peptide” or “protein” refers to a compound oftwo or more amino acids in which the carboxyl group of one is unitedwith an amino group of another, forming a peptide bond. The term peptideis also used to denote the amino acid sequence encoding such a compound.As used herein, “isolated” or “synthesized” peptide or biologicallyactive portion thereof refers to a peptide that is substantially free ofcellular material or other contaminating peptides from the cell ortissue source from which the peptide is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesizedby any method, or substantially free from contaminating peptides whensynthesized by recombinant gene techniques.

As used herein, a Replikin peptide or Replikin protein is an amino acidsequence having 7 to about 50 amino acids comprising:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue;    -   (3) at least 6% lysine residues.        Similarly, a Replikin sequence is the amino acid sequence        encoding such a peptide or protein.

As used herein, “emerging strain” as used herein refers to a strain of avirus, bacterium, fungus, or other organisms identified as having anincreased increasing concentration of Replikin sequences in one or moreof its protein sequences relative to the concentration of Replikins inother strains of such organism. The increase or increasing concentrationof Replikins occurs over a period of at least about six months, andpreferably over a period of at least about one year, most preferablyover a period of at least about three years or more, for example, ininfluenza virus, but may be a much shorter period of time for bacteriaand other organisms.

As used herein, “mutation” refers to change in this structure andproperties of an organism caused by substitution of amino acids. Incontrast, the term “conservation” as used herein, refers to conservationof particular amino acids due to lack of substitution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting the frequency of occurrence of Replikinsin various organisms.

FIG. 2 is a graph depicting the percentage of malignin per milligramtotal membrane protein during anaerobic replication of glioblastomacells.

FIG. 3 is a bar graph showing amount of antimalignin antibody producedin response to exposure to the recognin 16-mer.

FIG. 4A is a photograph of a blood smear taken with ordinary andfluorescent light. FIG. 4B is a photograph of a blood smear taken withordinary and fluorescent light illustrating the presence of two leukemiccells. FIG. 4C is a photograph of a dense layer of glioma cells in thepresence of antimalignin antibody. FIG. 4D and FIG. 4E are photographsof the layer of cells in FIG. 4C taken at 30 and 45 minutes followingaddition of antimalignin antibody

FIG. 4F is a bar graph showing the inhibition of growth of small celllung carcinoma cells in vitro by antimalignin antibody.

FIG. 5 is a plot of the amount of antimalignin antibody present in theserum of patients with benign or malignant breast disease pre- and postsurgery.

FIG. 6 is a box diagram depicting an embodiment of the invention whereina computer is used to carry out the 3-point-recognition method ofidentifying Replikin sequences.

FIG. 7 is a graph showing the concentration of Replikins observed inhemagglutinin of influenza B and influenza A strain, H1N1, on a year byyear basis from 1918 through 2001.

FIG. 8 is a graph of the Replikin concentration observed inhemagglutinin of influenza A strains, H2N2 and H3N2, as well as anemerging strain defined by its constituent Replikins, designatedH3N2(R), on a year by year basis from 1950 to 2001.

DETAILED DESCRIPTION OF THE INVENTION

The identification of a new family of small peptides related to thephenomenon of rapid replication, referred to herein as Replikins,provides targets for detection of pathogens in a sample and developingtherapies, including vaccine development. In general, knowledge of andidentification of this family of peptides enables development ofeffective therapies and vaccines for any organism that harborsReplikins. Identification of this family of peptides also provides forthe detection of viruses and virus vaccine development.

For example, identification of this family of peptides provides for thedetection of influenza virus and provides new targets for influenzatreatment. Identification of this family of peptides also provides forexample, for the detection of malaria and provides new targets formalaria vaccine development. Further examples provided by theidentification of this family of peptides include the detection ofinfectious disease Replikins, cancer immune Replikins and structuralprotein Replikins.

Rapid replication is characteristic of virulence in certain bacteria,viruses and malignancies, but no chemistry common to rapid replicationin different organisms has been described. We have found a family ofconserved small protein sequences related to rapid replication, which wehave named Replikins. Such Replikins offer new targets for developingeffective detection methods and therapies. The first Replikin found wasthe glioma Replikin, which was identified in brain glioblastomamultiforme (glioma) cell protein called malignin.

Hydrolysis and mass spectrometry of malignin revealed the novel 16 merpeptide sequence which contains the glioma Replikin. This Replikin wasnot found in databases for the normal healthy human genome and thereforeappeared to be derived from some source outside the body.

We have devised an algorithm to search for the glioma Replikin orhomologue thereof Homologues were not common in over 4,000 proteinsequences, but were found, surprisingly, in all tumor viruses, and inthe replicating proteins of algae, plants, fungi, viruses and bacteria.

We have identified that both 1) Replikin concentration (number ofReplikins per 100 amino acids) and 2) Replikin composition correlatewith the functional phenomenon of rapid replication. These relationshipsprovide functional basis for the determination that Replikins arerelated quantitatively as well as qualitatively to the rate ofreplication.

The first functional basis for Replikins role to rapid replication isseen in glioma replication. The fact that glioma malignin is enrichedten-fold compared to the five-fold increase in cell number and membraneprotein concentration in rapid replication of glioma cells suggests anintegral relationship of the Replikins to replication. When the gliomaReplikin was synthesized in vitro and administered as a syntheticvaccine to rabbits, abundant antimalignin antibody was produced. Thisestablishes the antigenic basis of the antimalignin antibody in serum(AMAS) test, and provides the first potential synthetic cancer vaccineand the prototype for Replikin vaccines in other organisms. With thedemonstration of this natural immune relationship of the Replikins toreplication and this natural immune response to cancer Replikins, whichoverrides cell type, based upon the shared specificity of cancerReplikins and rapid replication, both passive augmentation of thisimmunity with antimalignin antibody and active augmentation withsynthetic Replikin vaccines now is possible.

The relationship between the presence of antimalignin antibody andsurvival in patients was shown in a study of 8,090 serum specimens fromcancer patients. The study showed that the concentration of antimaligninantibody increases with age, as the incidence of cancer in thepopulation increases, and increases further two to three-fold in earlymalignancy, regardless of cell type. In vitro, the antimalignin antibodyis cytotoxic to cancer cells at picograms (femtomoles) per cancer cell,and in vivo the concentration of antimalignin antibody relatesquantitatively to the survival of cancer patients. As shown in gliomacells, the stage in cancer at which cells have only been transformed tothe immortal malignant state but remain quiescent or dormant, now can bedistinguished from the more active life-threatening replicating state,which is characterized by the increased concentration of Replikins. Inaddition, clues to the viral pathogenesis of cancer may be found in thefact that glioma glycoprotein 10B has a 50% reduction in carbohydrateresidues when compared to the normal 1OB. This reduction is associatedwith virus entry in other instances, and so may be evidence of theattachment of virus for the delivery of virus Replikins to the 10B ofglial cells as a step in the transformation to the malignant state.

Our study concerning influenza virus hemagglutinin protein sequences andinfluenza epidemiology over the past 100 years, has provided a secondfunctional basis for the relations of Replikins to rapid replication.Only serological hemagglutinin and antibody classification, but nostrain-specific conserved peptide sequences have previously beendescribed in influenza. Further, no changes in concentration andcomposition of any strain-specific peptide sequences have been describedpreviously that correlate with epidemiologically documented epidemics orrapid replication. In this study, a four to ten-fold increase in theconcentration of strain-specific influenza Replikins in one of each ofthe four major strains, influenza B, (A)H1N1, (A)H2N2 and (A)143N2, isshown to relate to influenza epidemics caused by each strain from 1902to 2001.

We then showed that these increases in concentration are due to thereappearance of at least one specific Replikin composition from 1 to upto 64 years after its disappearance, plus the emergence of newstrain-specific Replikin compositions. Previously, no strain-specificchemical structures were known with which to predict the strains thatwould predominate in coming influenza seasons, nor to devise annualmixtures of whole-virus strains for vaccines. The recent sharp increasein H3N2 Replikin concentration (1997 to 2000), the largest in H3N2'shistory, and the reappearance of specific Replikin compositions thatwere last seen in the high mortality H3N2 pandemic of 1968, and in thetwo high mortality epidemics of 1975 and 1977, but were absent for 20-25years, together may be a warning of coming epidemics. This high degreeof conservation of Replikin structures observed, whereby the identicalstructure can persist for 100 years, or reappear after an absence offrom one to 64 years, indicate that what was previously thought to bechange due to random substitution of amino acids in influenza proteinsis more likely to be change due to an organized process of conservationof Replikins.

The conservation of Replikins is not unique to influenza virus but wasalso observed in other sources, for example in foot and mouth diseasevirus, type O, HIV tat, and wheat.

A third functional basis for Replikins' role in rapid replication isseen in the increase in rapid replication in HIV. Replikin concentrationwas shown to be related to rapid replication in HIV. We found theReplikin concentration in the slow growing low-titre strain of HIV (NS1,“Bru”), which is prevalent in early stage infection, to be one-sixth ofthe Replikin concentration in the rapidly-growing high-titre strain ofHIV (SI, “Lai”)(prevalent in late stage HIV infection).

Further examples demonstrate the relationship of Replikins to rapidreplication. In the “replicating protein,” of tomato curl leaf geminivirus which devastates tomato crops, the first 161 amino acids, thesequence which has been shown to bind to DNA, was shown to contain fiveReplikins In malaria, legendary for rapid replication when trypanosomesare released from the liver in the tens of thousands from onetrypanosome, multiple, novel, almost ‘flamboyant’ Replikin structureshave been found with concentrations of up to 36 overlapping Replikinsper 100 amino acids.

The conservation of any structure is critical to whether that structureprovides a stable invariant target to attack and destroy or tostimulate. When a structure is tied in some way to a basic survivalmechanism of the organism, the structures tend to be conserved. Avarying structure provides an inconstant target, which is a goodstrategy for avoiding attackers, such as antibodies that have beengenerated specifically against the prior structure and thus areineffective against the modified form. This strategy is used byinfluenza virus, for example, so that a previous vaccine may be quiteineffective against the current virulent virus.

Replikins as Stable Targets for Treatment

Both bacteria and HIV have both Replikin and non-Replikin amino acids.In HIV, for example, there has been a recent increase in drug-resistancefrom 9% to 13% due to mutation, that is substitution of non-Replikinamino acids. (See detailed analysis of TAT protein of HIV discussedherein). In bacteria, the development of ‘resistant strains’ is due to asimilar mechanism. However, we have found that Replikin structures donot mutate or change to the same degree as non Replikin amino acids (seealso discussion of foot and mouth disease virus conservation ofReplikins discussed herein). The Replikin structures, as opposed to thenon-Replikin structures are conserved and thus provide new constanttargets for treatment.

Certain structures too closely related to survival functions apparentlycannot change constantly. Because an essential component of the Replikinstructure is histidine (h), which is know for its frequent binding tometal groups in redox enzymes and probable source of energy needed forreplication, and since this histidine structure remains constant, thisstructure remains all the more attractive a target for destruction orstimulation.

From a proteomic point of view, inventors construction of a templatebased on the newly determined glioma peptide sequence led them to thediscovery of a wide class of proteins with related conserved structuresand a particular function, in this case replication. Examples of theincrease in Replikin concentration with virulence of a disease include,influenza, HIV, cancer and tomato leaf curl virus. This newly recognizedclass of structures is related to the phenomenon of rapid replication inorganisms as diverse as yeast, algae, plants, the gemini curl leaftomato virus, HIV and cancer.

Replikin concentration and composition provide new quantitative methodsto detect and control the process of replication, which is central tothe survival and dominance of each biological population. The sharing ofimmunological specificity by diverse members of the class, asdemonstrated with antimalignin antibody for the glioma and relatedcancer Replikins, suggests that B cells and their product antibodies mayrecognize Replikins by means of a similar recognition language.

Examples of peptide sequences of cancer Replikins or as containing aReplikin, i.e., a homologue of the glioma peptide, kagvaflhkk, may befound in such cancers of, but not limited to, the lung, brain, liver,soft-tissue, salivary gland, nasopharynx, esophagus, stomach, colon,rectum, gallbladder, breast, prostate, uterus, cervix, bladder, eye,forms of melanoma, lymphoma, leukemia, and kidney.

Replikins provide for: 1) detection of pathogens by qualitative andquantitative determinations of Replikins; 2) treatment and control of abroad range of diseases in which rapid replication is a key factor bytargeting native Replikins and by using synthetic Replikins as vaccines;and 3) fostering increased growth rates of algal and plant foods.

The first Replikin sequence to be identified was the cancer cellReplikin found in a brain cancer protein, malignin, which wasdemonstrated to be enriched ten-fold during rapid anaerobic replicationof glioblastoma multiforme (glioma) cells. (FIG. 2) Malignin is a 10 KDaportion of the 250 KDa glycoprotein 10B, which was isolated in vivo andin vitro from membranes of glioblastoma multiforme (glioma) cells.Hydrolysis and mass spectroscopy of malignin revealed a 16-mer peptidesequence, ykagvaflhkkndide (SEQ ID NO.:4), which is referred to hereinas the glioma Replikin and which includes the shorter peptide,kagvaflhkk (SEQ ID NO.: 1), both of which apparently are absent in thenormal human genome.

TABLE 1 16-mer peptide sequence ykagvaflhkkndide obtained from maligninby hydrolysis and mass spectrometry Method By Which Fragment ObtainedAuto- hydrolysis of malignin Auto- immobilized Seq hydrolysis of on IDFragment MH+ malignin free bromoace-tyl Microwaved Microwaved NO.Identified (mass) Seqence in solution cellulose 5 seconds 30 seconds 191-3 381.21 ( )yka(g) + 20 1-5 537.30 ( )ykagv(a) + 21 2-6 445.28(y)kagva(f) + 22 2-7 592.35 (Y)kagvaf(l) + 23  4-11 899.55(a)gvaflhkk(n) + 24 5-7 336.19 (g)vaf(l) + 25 6-7 237.12 (v)af(l) + 26 6-10 615.36 (v)aflhk(k) + 27  6-10 615.36 (v)aflhk(k) + 28  6-12 857.50(v)aflhkkn(d) + 29  6-12 857.50 (v)afhkkn(d) + 30 7-8 279.17 (a)fl(h) +31 10-16 861.43 (h)kkndide( ) + 32 11-14 489.27 (k)kndi(d) + 33 12-15476.2 (k)ndid(e) +

When the 16-mer glioma Replikin was synthesized and injected as asynthetic vaccine into rabbits, abundant antimalignin antibody wasproduced. (Bogoch et al., Cancer Detection and Prevention, 26 (Suppl.1): 402 (2002)). The concentration of antimalignin antibody in serum invivo has been shown to relate quantitatively to the survival of cancerpatients. (Bogoch et al., Protides of Biological Fluids, 31:739-747(1984). In vitro antimalignin antibodies have been shown to be cytotoxicto cancer cells at a concentration of picograms (femtomolar) per cancercell. (Bogoch et al., Cancer Detection and Prevention, 26 (Suppl. 1):402 (2002).

Studies carried out by the inventors showed that the glioma Replikin isnot represented in the normal healthy human genome. Consequently, asearch for the origin and possible homologues of the Replikin sequencewas undertaken by analysis of published sequences of various organisms.

By using the 16-mer glioma a Replikin sequence as a template andconstructing a recognition proteomic system to visually scan the aminoacid sequences of proteins of several different organisms, a new classof peptides, the Replikins, was identified. The present inventionprovides a method for identifying nucleotide or amino acid sequencesthat include a Replikin sequence. The method is referred to herein as a3-point-recognition method. The three point recognition methodcomprises: a peptide from 7 to about 50 amino acids including:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues. (Replikin).        These peptides or proteins constitute a new class of peptides in        species including algae, yeast, fungi, amoebae, bacteria, plant,        virus and cancer proteins having replication, transformation, or        redox functions. Replikin peptides have been found to be        concentrated in larger ‘replicating’ and ‘transforming’ proteins        (so designated by their investigators, See Table 2) and cancer        cell proteins. No sequences were found to be identical to the        malignin 16-mer peptide.

TABLE 2 Examples of Replikins in various organisms - prototype: GliomaReplikin* kagvaflhkk (SEQ ID No.: 1) SEQ ID NO. Algae: 34 Caldopheraprolifera kaskftkh 35 Isolepisprolifera kaqaetgeikgh Yeast: 36Schizosaccharomyces pombe ksfkypkkhk 37 Oryza sativa kkaygnelhk  2Sacch. cerevisiae replication binding protein hsikrelgiifdk Fungi: 38Isocitrate lyase ICI I, Penicillium marneffei kvdivthqk 39 DNA-dependentRNA polymerase II, Discula destructiva klcedaayhrkk 40 Ophiostomanovo-ulm I, RNA in Dutch elm disease kvilplrgnikgiffkh fungus Amoeba: 41Entamoeba invadens, histone H2B klilkgdlnkh Bacteria: 42 Pribosomalprotein replication factor, Helicobacter pylori ksvhaflk 10Replication-associated protein Staph. Aureus 43 Mycoplasma pulmonic,chromosome replication kkcktthnk 90 Macrophage infectivity potentiator,L. legionella kvhffqlkk Plants: 44 Arabidopsis thaliana, proliferakdhdfdgdk 45 Arabidopsis thaliana, cytoplasmic ribosomal kmkglkqkkah 46Arabidopsis thaliana, DNA binding protein kelssttqeksh Viruses:  9Replication associated protein A [Maize streak virus] Kekkpskdeimrdiish11 Bovine herpes virus 4, DNA replication protein hkinitngqk 12Meleagrid herpesvirus I, replication binding protein hkdlyrllmk 47Feline immunodeficiency hlkdyklvk  3 Foot and Mouth Disease (O)hkqkivapvk  5 HIV Type 1 kcfncgkegh  7 HIV Type 2 kcwncgkegh Tumor 48Rous sarcoma virus tyrosine-protein kinase kklrhek Viruses: 49 v-yes,avian sarcoma kklrhdk 50 c-yes, colon cancer, malignant melanoma kklrhdk51 v-srcC, avian sarcoma kklrhek 52 c-src, colon, mammary, panrcreaticcancer kklrhek 53 Neuroblastoma RAS viral (v-ras) oncogene kqahelak 54VPI (major capsid protein) [Polyamavirus sp.] kthrfskh 55 Sindbisknlhekik 56 E1 [Human papilloamavirus type 71] khrpllqlk 57 v-erbB fromAEV and c-erb kspnhvk 58 v-fms (feline sarcoma) knihlekk 59 c-fms (acuteand chronic myclomonocytic tumors) knihlekk 60 large t-antigen I[Polyomavirus sp.] kphlaqslek 61 middle t-antigen [Polyomavirus sp,]-kqhrelkdk 62 small t-antigen [Polyomavirus sp], kqhrelkdk 63 v-abl,munine acute leukemia kvpvlisptlkh 64 Human T-cell lymphotropic virustypo 2 kslllevdkdish 65 c-kit, GI tumors, small cell lung carcinomakagitimvkreyh 18 Hepatitis C hyppkpgcivpak Trans- 66 Transformingprotein myb Ksgkhlgk Forming 67 Transforming protein myc, Burkittlymphoma krreqlkhk Proteins: 68 Ras-related GTP-binding proteinksfevikvih 69 Transforming protein ras (teratocarcinoma) kkkhtvkk 70TRAF-associated NF•kB activator TANK kaqkdhlsk 71 RFP transformingprotein hlkrvkdlkk 72 Transforming protein D (S.C.) kygspkhrlik 73Papilloma virus type II, transforming protein klkhilgkarfik 74 Proteintryosine kinase (BC 2.7.1.112slk kgdhvkhykirk 75 Transforming protein(axl(-)) keklrdvmvdrhk 76 Transforming protein (N-myc) klqarqqqllkkieh77 Fibroblast growth factor 4 (Kaposi sarcoma) kkgnrvsptmkvth Cancer 78Matrix metaloproteinase 7 (uterine) keiplhfrk Cell 79 Transcriptionfactor 7-like kkkphikk Proteins: 80 Breast cancer antigen NY-BR-87ktrhdplak 81 BRCA-1-Associated Ring Domain Protein (breast) khhpkdnlik82 ‘Autoantigen from a breast tumor’ khkrkkfrqk 83 Glioma Replikin (thisstudy) kagvaflhkk 84 Ovarian cancer antigen khkrkkfrqk 85 EE L leukemiakkkskkhkdk 86 Proto-oncogene tyrosine-protein kinase C-ABLE hksckpalprk87 Adenomatosis polyposis coli kkkkpsrlkgdnek 88 Gastric cancertransforming protein ktkkgnrvsptmkvth 89 Transforming protein (K-RAS2B),lung khkekmskdgkkkkkksk

Identification of an amino acid sequence as a Replikin or as containinga Replikin, i.e., a homologue of the glioma peptide, kagvaflhkk,requires that the three following requirements be met. According to thethree point recognition system the sequences have three elements: (1) atleast one lysine residue located six to ten residues from another lysineresidue; (2) at least one histidine residue; and (3) a composition of atleast 6% lysine within an amino acid sequence of 7 to about 50 residues.

Databases were searched using the National Library of Medicine keyword“PubMed” descriptor for protein sequences containing Replikin sequences.Over 4,000 protein sequences were visually examined for homologues.Sequences of all individual proteins within each group ofPubMed-classified proteins were visually scanned for peptides meetingthe three above-listed requirements. An infrequent occurrence ofhomologues was observed in “virus peptides” as a whole (1.5%) (N=953),and in other peptides not designated as associated with malignanttransformation or replication such as “brain peptides” and“neuropeptides” (together 8.5%) (N=845). However, surprisingly,homologues were significantly more frequently identified in large“replicating proteins,” which were identified as having an establishedfunction in replication in bacteria, algae, and viruses. Even moresurprising was the finding that Replikin homologues occurred in 100% of“tumor viruses” (N=250), in 97% of “cancer proteins” (N=401), and in 85%of “transforming viruses” (N=248). These results suggest that there areshared properties of cancer pathogenesis regardless of cell type andsuggest a role of viruses in carcinogenesis, i.e., conversion of cellsfrom a transformed albeit dormant state to a more virulent activelyreplicating state.

Homologues of the following amino acid sequence, kagvaflhkk, as definedby the three point recognition method, were found in such viruses, orviral peptides, as, but not limited to, adenovirus, lentivirus, a-virus,retrovirus, adeno-associated virus, human immunodeficiency virus,hepatitis virus, influenza virus, maize streak virus, herpes virus,bovine herpes virus, feline immunodeficiency virus, foot and mouthdisease virus, small pox virus, rous sarcoma virus, neuroblastoma RASviral oncogene, polyamavirus, sindbis, human papilloma virus,myelomonocytic tumor virus, murine acute leukemia, T-cell lymphotropicvirus, and tomato leaf curl virus.

Replikins are present in such bacteria as, but not limited to,Acetobacter, Achromobacter, Actinomyces, Aerobacter, Alcaligenes,Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Chainia,Clostridium, Corynebacterium, Erwinia, Escheria, Lebsiella,Lactobacillus, Haemophilus, Flavobacterium, Methylomonas, Micrococcus,Mycobacterium, Micronomspora, Mycoplasma, Neisseria, Nocardia, Proteus,Pseudomonas, Rhizobium, Salmonella, Serratia, Staphylococcus,Streptocossus, Streptomyces, Streptosporangium, Streptovirticillium,Vibrio, peptide, and Xanthomas.

Replikins are present in such fungi as, but not limited to, Penicillium,Diseula, Ophiostoma novo-ulim, Mycophycophta, Phytophthora infestans,Absidia, Aspergillus, Candida, Cephalosporium, Fusarium, Hansenula,Mucor, Paecilomyces, Pichia, Rhizopus, Torulopsis, Trichoderma, andErysiphe.

Replikins are present in such yeast as, but not limited to,Saccharomyces, Cryptococcus, including Cryptococcus neoformas,Schizosaccharomyces, and Oryza.

Replikins are present in algae such as, but not limited to, Caldophera,Isolepisprolifera, Chondrus, Gracilaria, Gelidium, Caulerpa, Laurencia,Cladophexa, Sargassum, Penicillos, Halimeda, Laminaria, Fucus,Ascophyllum, Undari, Rhodymenia, Macrocystis, Eucheuma, Ahnfeltia, andPteroclasia.

Replikins are present in amoeba such as, but not limited to, Entamoeba(including Entamoeba invadens), Amoebidae, Acanthamoeba and Naegleria.

Replikins are present in plants such as, but not limited to,Arabidopsis, wheat, rice, and maize.

Auxiliary Specifications

To permit classification of subtypes of Replikins, additional or“auxiliary specifications” to the basic “3-point-recognition”requirements may be added: (a) on a structural basis, such as the commonoccurrence of adjacent di- and polylysines in cancer cell proteins(e.g., transforming protein P21B(K-RAS 2B), lung, Table 2, SEQ ID NO.:89), and other adjacent di-amino acids in TOLL-like receptors, or b) ona functional basis, such as exhibiting ATPase, tyrosine kinase or redoxactivity as seen in Table 2.

Functional Derivatives

“Functional derivatives” of the Replikins as described herein arefragments, variants, analogs, or chemical derivatives of the Replikins,which retain at least a portion of the immunological cross reactivitywith an antibody specific for the Replikin. A fragment of the Replikinpeptide refers to any subset of the molecule. Variant peptides may bemade by direct chemical synthesis, for example, using methods well knownin the art. An analog of a Replikin to a non-natural proteinsubstantially similar to either the entire protein or a fragmentthereof. Chemical derivatives of a Replikin contain additional chemicalmoieties not normally a part of the peptide or peptide fragment.

As seen in FIG. 2, during anaerobic respiration when the rate of cellreplication is increased, malignin is enriched. That is, malignin isfound to increase not simply in proportion to the increase in cellnumber and total membrane proteins, but is enriched as much as ten-foldin concentration, starting with 3% at rest and reaching 30% of totalmembrane protein. This clear demonstration of a marked increase inReplikin concentration with glioma cell replication points to, and isconsistent with, the presence of Replikins identified with the 3-pointrecognition method in various organisms. For example, Replikins wereidentified in such proteins as “Saccharomyces cerevisiae replicationbinding protein” (SEQ ID NO.: 2) (hsikrelgiifdk); the “replicationassociated protein A of maize streak virus” (SEQ ID NO.: 8)(kyivcarealtk) and (SEQ ID NO.: 9) (kekkpskdeimrdiish); the“replication-associated protein of Staphylococcus aureus” (SEQ ID NO.:10) (kkektthnk); the “DNA replication protein of bovine herpes virus 4”(SEQ ID NO.: 11) (hkinitngqk); and the “Mealigrid herpes virus 1replication binding protein” (SEQ ID NO.: 12) (hkdlyrllmk). Previousstudies of tomato leaf curl gemini virus show that the regulation ofvirus accumulation appears to involve binding of amino acids 1-160 ofthe “replicating protein” of that virus to leaf DNA and to otherreplication protein molecules during virus replication. Analysis of thissequence showed that amino acids 1-163 of this “replicating protein”contain five Replikins, namely: (SEQ ID NO.: 13) kfrinaknyfltyph, (SEQID NO.: 14) knletpvnklfiricrefh, (SEQ ID NO.: 15) hpniqaaksstdvk, (SEQID NO.: 16) ksstdvkaymdkdgdvldh, and (SEQ ID NO.: 17)kasalnilrekapkdfvlqfih.

Table 2 shows that Replikin-containing proteins also are associatedfrequently with redox functions, and protein synthesis or elongation, aswell as with cell replication. The association with metal-based redoxfunctions, the enrichment of the Replikin-containing glioma maligninconcentration during anaerobic replication, and the cytotoxicity ofantimalignin at low concentrations (picograms/cell) (FIG. 4 c-f), allsuggest that the Replikins are related to central respiratory survivalfunctions, have been found less often subjected to the mutationscharacteristic of non-Replikin amino acids.

Of particular interest, it was observed that at least one Replikin per100 amino acids was found to be present in the hemagglutinin proteins ofalmost all of the individual strains of influenza viruses examined. TheReplikin sequences that were observed to occur in the hemagglutininproteins of isolates of each of the four prevalent strains of influenzavirus, influenza B, H1N1, H2N2, and H3N2, for each year that amino acidsequence data are available (1902-2001), are shown in Tables 3, 4, 5 and6, below.

TABLE 3 Replikin Sequences present in hemagglutinins of Influenza Bviruses in each year for which amino acid sequences were available(1902-2001). Influenza B Replikins Year Detected in Influenza B strain(Peak in FIG. 7: EB1 EB2) kshfanlk 1902, 19, 24, 38, 40, 43, 51, 59, 75,76, 77, 89, 90, 93, 97, 98, 99, (SEQ ID NO. 91) 00, 01 kshfanlkgtk 1902,19, 24, 38, 40, 43, 51, 59, 75, 76, 77, 89, 90, 93, 97, 98, 99, (SEQ IDNO. 92) 00, 01 kshfanlkgtktrgklcpk 1902, 19, 24, 38, 40, 43, 51, 59, 75,76, 77, 89, 90, 93, 97, 98, 99, (SEQ ID NO. 93) 00, 01 hekygglnk 1902,19, 24, 38, 40, 43, 51, 59, 75, 76, 77, 89, 90, 93, 97, 98, 99, (SEQ IDNO. 94) 00, 01 hekygglnksk 1902, 19, 24, 38, 40, 43, 51, 59, 75, 76, 77,89, 90, 93, 97, 98, 99, (SEQ ID NO. 95) 00, 01 hekygglnkskpyytgehak1902, 19, 24, 38, 40, 43, 51, 59, 75, 76, 77, 89, 90, 93, 97, 98, 99,(SEQ ID NO. 96) 00, 01 hakaigncpiwvk 1902, 19, 24, 38, 40, 43, 51, 59,75, 76, 77, 89, 90, 93, 97, 98, 99, (SEQ ID NO. 97) 00, 01hakaigncpiwvktplklangtk 1902, 19, 24, 38, 40, 43, 51, 59, 75, 76, 77,89, 90, 93, 97, 98, 99, (SEQ ID NO. 98) 00, 01hakaigncpiwvktplklangtkyrppak 1902, 19, 24, 38, 40, 43, 51, 59, 75, 76,77, 89, 90, 93, 97, 98, 99, (SEQ ID NO. 99) 00, 01hakaigncpiwvktplklangtkyrppakllk 1902, 19, 24, 38, 40, 43, 51, 59, 75,76, 77, 89, 90, 93, 97, 98, 99, (SEQ ID NO. 100) 00, 01 hfanlkgtktrgk1919, 76, 89, 90, 99, 00, 01 (SEQ ID NO. 101) hfanlkgtktrgklcpk 1919,76, 90 00, 01 (SEQ ID NO. 102) hsdneiqmvklygdsk 1919 (SEQ ID NO. 103)hsdneiqdkmvklygdskpqk 1919 (SEQ ID NO. 104) hsdneiqmvklygdskpqk 1919,24, 97, 98, 00 (SEQ ID NO. 105) k(a/v)silhevk 1919, 40, 59, 90, 93 (SEQID NO. 106) kctgtipsakasilh 1919, 00 (SEQ ID NO. 107) kctgtipsakasilhevk1919, 93 (SEQ ID NO. 108) kygglnkskpyytgeh 1919 (SEQ ID NO. 109)kvwcasgrskvikgslpligeadclh 1919, 38, 40, 43, 59, 75, 76, 77, 89, 90, 98,99, 00 (SEQ ID NO. 110) kpyytgehak 1919, 38, 40, 59, 89, 90, 93, 97, 98,01 (SEQ ID NO. 111) kcmgtipsakasilhevk 1924, 43, 75, 76, 77, 93 (SEQ IDNO. 112) hnvinaekapggpyk 1938, 93, 97, 00 (SEQ ID NO. 113)hsdnetqmaklygdsk 1938, 93, 97, 00 (SEQ ID NO. 114) hgvavaadlkstqeaink1940, 59, 00 (SEQ ID NO. 115) hgvavaadlkstqeainkdtistqeaink 1940 (SEQ IDNO. 116) klygdskpqkftssangvtth 1943, 75, 76, 77, 93, 97, 00 (SEQ ID NO.117) hsdnetqmaklygdskpqk 1943, 75, 76, 77, 93 (SEQ ID NO. 118)hfanlkgtqtrgk 1959 (SEQ ID NO. 119) kprsalkckgfh 1988 (SEQ ID NO. 120)kskpyytgehakai(g/a)ncpiwvk 2000 (SEQ ID NO. 121) 1. Influenza B has notbeen responsible for any human pandemic (global distribution). 2.Abbreviation for years: eg. “19”=1919, “01”=2001. 3. The first year thata given Replikin appears is indicated at the beginning of the series ofyears in which that Replikin has been found. 4. Overlapping Replikinsequences are listed separately. 5. Increase in number of new Replikinstructures occurs in years of epidemics (underlined): eg. 1951 and 1977and correlates with increased total Replikin concentration (number ofReplikins per 100 amino acid residues). See FIG. 7.

TABLE 4 H1N1 Replikin Sequences present in H1N1 hemagglutinins ofInfluenza viruses in each year for which amino acid sequences wereavailable (1918-2000) H1N1 Replikin Year Detected in Influenza H1N1Strain Peak in FIG. 7 P1 E1 E1.1, 1.2, 1.3 E1.4)hp(v/t)tigecpkyv(r/k)(s/t)(t/a)k 1918, 25, 28, 30, 31, 35, 47, 48, 51,(SEQ ID NO. 122) 52, 55, 56, 57, 59, 63, 77, 79, 80, 81, 85, 87, 88, 89,91, 92, 95, 96, 97, 98, 99, 00 hdsnvknly(e/g)kv(k/r)(n/s)ql(k/r)nnak1918, 28, 30, 31, 77, 79, 80, 88, 91, (SEQ ID NO. 123) 95, 98hdsnvknly(e/g)kv(k/r)(n/s)qlk 1918, 28, 30, 31, 77, 79, 80, 88, 91, (SEQID NO. 124) 95, 98hkc(nn/dd)(a/t/e)cmesv(r/k)ngtydypkyseesklnre(e/k)idgvk 1918, 30, 35,77, 80, 98 (SEQ ID NO. 125) hkc(nn/dd)(a/t/e)cmesv(r/k)ngtydypkyseesk1918, 30, 35, 77, 80, 98 (SEQ ID NO. 126)hqn(e/g)qgsgyaadqkstqnai(d/n)gitnkvnsviekmntqftavgkefnklek 1918, 28, 30,31, 35, 59, 79, 95 (SEQ ID NO. 127)hqn(e/g)qgsgyaadqkstqnai(d/n)gitnkvnsviek 1918, 28, 30, 31, 35, 59, 79,95 (SEQ ID NO. 128) hqn(e/g)qgsgyaadqkstqnai(d/n)gitnk 1918, 28, 30, 31,35, 59, 79, 95 (SEQ ID NO. 129) kfeifpktsswpnh 1918, 77 (SEQ ID NO. 130)kg(n/s/t)sypkl(n/s)ksy(v/t)nnkgkevlvlwgvh 1918, 35, 77, 96 (SEQ ID NO.131) ksy(v/t)nnkgkevlvlwgvh 1918, 35, 77, 96 (SEQ ID NO. 132)hkenneemesvkngtydypkyseesklnrekidgvk 1928, 31, 95 (SEQ ID NO. 133)hkenneemesvkngtydypkyseesk 1928, 31, 95 (SEQ ID NO. 134)hkenneemesvkngtydypk 1928, 31, 95 (SEQ ID NO. 135) hkennecmesvk 1928,31, 95 (SEQ ID NO. 136) hngkssfy(k/r)nllwlt(e/g)knglypnlsksyvnnkek 1928,95, 00 (SEQ ID NO. 137) hngkssfy(k/r)nllwlt(e/g)knglypnlsksyvnnk 1928,31, 95, 00 (SEQ ID NO. 138) hngkssfy(k/r)nllwlt(e/g)knglypnlsk 1928, 31,95, 00 (SEQ ID NO. 139) hngkssfy(k/r)nllwlt(e/g)k 1928, 31, 95, 00 (SEQID NO. 140) kssfyknllwlteknglypnlsksyvnnkekevlvlwgvh 1928, 31, 95 (SEQID NO. 141) knllwlteknglypnlsksyvnnkekevlvlwgvh 1928, 31, 95 (SEQ ID NO.142) knglypnlsksyvnnkekevlvlwgvh 1928, 31, 95, 96, 00 (SEQ ID NO. 143)ksy(v/a)nnkekev(l/-)(v/-)lwgvh 1928, 31, 51, 95, 96, 98, 00 (SEQ ID NO.144) kesswpnhtvtk 1928, 31, 95 (SEQ ID NO. 145)het(t/n)kgvtaacpyagassfyrnllwlvkkensypklsksyvnnk 1930, 35 (SEQ ID NO.146) het(t/n)kgvtaacpyagassfymllwlvkkensypklsk 1930, 35 (SEQ ID NO. 147)kfeifpktsswpnevlvlwgvh 1930 (SEQ ID NO. 148) kerswpkh 1947, 51, 52, 55,56, 79, 82 (SEQ ID NO. 149) klsksyvnnkekevlvlwqvh 1947, 51 (SEQ ID NO.150) knnkekevlvlwqvh 1947 (SEQ ID NO. 151)h(k/n)(g/q)kssfy(r/k)nllwltekng(l/s)yp(n/t)lsksyannkek 1948 79, 89, 96(SEQ ID NO. 152) h(k/n)(g/q)kssfy(r/k)nllwltek 1948 79, 89, 96 (SEQ IDNO. 153) hakkssfyk 1951, 57, 59 (SEQ ID NO. 154) hngklcrlkgk 1951, 52,55, 56, 57, 59, 79 (SEQ ID NO. 155) hyklnn(q/g)kk 1956, 00 (SEQ ID NO.156) hdiyrdeainnrfqiqgvkltqgyk 1956 (SEQ ID NO. 157) kgngcfeifhk 1956(SEQ ID NO. 158) klnrliektndkyhqiek 1956 (SEQ ID NO. 159) klnrliektndkyh1956 (SEQ ID NO. 160) kchtdkgslsttk 1956 (SEQ ID NO. 161)kinngdyaklyiwgvh 1956 (SEQ ID NO. 162) hngklcrkgiaplqlgk 1959, 82 (SEQID NO. 163) hetnrqvtaacpyagansffrnhiwlvkkessypklsk 1963, 81 (SEQ ID NO.164) hetnrqvtaacpyagansffrnliwlvkkessypk 1963, 81 (SEQ ID NO. 165)hpptstdqqslyqnadayifvgsskynrkfk 1963, 81 (SEQ ID NO. 166)hpptsrdqqslyqnadayifvgsskynrkfkpeia 1963, 81 (SEQ ID NO. 167)hdiyrdeainnrfqiqgvkitqgyk 1977, 79, 91 (SEQ ID NO. 168)hqneqgsgyaadqkstqnaidgitnkvnsviekmntqftavgk 1977 (SEQ ID NO. 169)hqneqgsgyaadqkstqnaidgitnkvnsviek 1977 (SEQ ID NO. 170)hqneqgsgyaadqkstqnaingitnkvnsviekmntqftavgkefnklek 1979, 91 (SEQ ID NO.171) hngklcrlkgiaplqlgk 1979 (SEQ ID NO. 172) hkcnnecmesvk 1979 (SEQ IDNO. 173) kfeifpkasswpnh 1981 (SEQ ID NO. 174) hdsnvknlyekvrsqlrnnak 1981(SEQ ID NO. 175) kvnsvikkmntqfaavgkefnh 1981 (SEQ ID NO. 176) khngklck1981 (SEQ ID NO. 177) kkgtsypklsksythnkgkevlvlwgvh 1981 (SEQ ID NO. 178)kgtsypklsksythnkgkevlvlwgvh 1981 (SEQ ID NO. 179) klsksythnkgkevlvlwgvh1981 (SEQ ID NO. 180) ksythnkgkevlvlwgvh 1981 (SEQ ID NO. 181)kgvtascshk 1985, 87 (SEQ ID NO. 182) kgvtascshkgrssfyrnllwlteknglypnlsk1985, 87 (SEQ ID NO. 183) kgnsypklsksyvnnkekevlvlwgih 1988 (SEQ ID NO.184) kefnhlek 1988 (SEQ ID NO. 185)hpptstdqqslyqnadayvfvgsskynkkfkpeiatrpk 1988 (SEQ ID NO. 186)hpptstdqqslyqnadayvfvgsskynkkfk 1988 (SEQ ID NO. 187)hegkssfyrnllwltekegsypklknsyvnk 1991 (SEQ ID NO. 188)hegkssfyrnllwltekegsypk 1991 (SEQ ID NO. 189) hkcdnecmesvrngtydypkyseesk1991 (SEQ ID NO. 190) kesswpnhtvtk 1991, 92 (SEQ ID NO. 191)knllwlteknglypnlsksyvnnkekeilvlwgvh 1991, 92, 96 (SEQ ID NO. 192)hngkssfy(k/m)(n/-)llwlt(e/g)(-/k)knglypnlsk 1991, 92, 96, 00 (SEQ ID NO.193) hngkssfyknllwltek 1991, 92, 96 (SEQ ID NO. 194)htvtkgvtascshngkssfyknllwlteknglypnlsksyvnnkekevlvlwgvh 1995 (SEQ ID NO.195) htvt(k/g)gv(t/s)ascshngkssfy(k/m)(n/-)llwlt(e/g)k(-n/k)glypnlsk1995, 00 (SEQ ID NO. 196) htvtkgvtascshngkssfyknllwltek 1995 (SEQ ID NO.197) kyvrstklrmvtglrnipsiqsrglfgaiagfieggwtgmidgwygyh 1995 (SEQ ID NO.198) hqneqgsgyaadqkstqnaingitnkvnsiiekmntqftavgk 1995 (SEQ ID NO. 199)hqneqgsgyaadqkstqnaingitnkvnsiiek 1995 (SEQ ID NO. 200)hqneqgsgyaadqkstqnaingitnk 1995 (SEQ ID NO. 201) hsgarsfyrnllwivkkgnsypk1996 (SEQ ID NO. 202) hsgarsfyrnllwivkkgnsypklnk 1996 (SEQ ID NO. 203)hsgarsfyrnllwivkkgnsypklnksytndk 1996 (SEQ ID NO. 204)hsgarsfyrnllwivkkgnsypklnksytndkgk 1996 (SEQ ID NO. 205)htvskgvttscshngk 1996 (SEQ ID NO. 206) katswpnhettk 1996 (SEQ ID NO.207) kqvttscshnqk 1996 (SEQ ID NO. 208) kgnsvpklnksytndkgkevlviwgvh 1996(SEQ ID NO. 209) klnksytndkgkevlviwgvh 1996 (SEQ ID NO. 210)ksytndkgkevlviwgvh 1996 (SEQ ID NO. 211)hnqkssfyrnllwlt(e/q)knglypnlsksy(v/a)annkek 1997, 98, 99 (SEQ ID NO.212) hpitigecpkyvrsak 1997 (SEQ ID NO. 213)hqneqgsgyaadqkstqnaingitnkvnsvlekmntqftavgk 1998 (SEQ ID NO. 214)hqneqgsgyaadqkstqnaingitnkvnsviek 1998 (SEQ ID NO. 215)hngkssfyrnllwlteknglypnlsksyvnnkek 1998 (SEQ ID NO. 216) 1. InfluenzaH1N1 was responsible for the human pandemic (global distribution) of1918. 2. Abbreviation for years: eg. “96” = 1996. 3. The first year thata given Replikin appears is indicated at the beginning of the series ofyears in which that Replikin has been found in this work. 4. OverlappingReplikin sequences are listed separately. 5. Increase in number of newReplikin structures occurs in years of epidemics (underlined): eg. 1918and 1977 and correlates with increased total Replikin concentration(number of Replikins per 100 amino acid residues). See FIG. 7.

TABLE 5 Replikin Sequences present in hemagglutinins of Influenza H2N2viruses in years 1957-2000 Influenza H2N2 Reylikins Year Detected inInfluenza H2N2 strain (Peak in FIG. 8: P2 E2) khfekvkilpk 1957, 58, 59,60, 61, 64, 65, 68, 78, 83, 84, 91 (SEQ ID NO. 217) khllssvkhfekvk 1957,58, 59, 60, 61, 83, 84, 91 (SEQ ID NO. 218) ha(k/q/m)(d/n)ilekthngk1957, 58, 59, 60, 61, 64, 65, 68, 78, 83, 84, 91, 95 (SEQ ID NO. 219)ha(k/q/m)(d/n)ilekthngklc(k/r) 1957, 58, 59, 60, 61, 64, 65, 68, 78, 83,84, 91, 95 (SEQ ID NO. 220) hnvhpltigecpkyvksek 1957, 58, 59, 65, 68(SEQ ID NO. 221) hpltigecpkyvksek 1957, 58, 59, 65, 68, 64, 65, 68, 78,83, 84, 91 (SEQ ID NO. 222) khllssvkhfekvkilpk 1957, 58, 59, 60, 61, 64,65, 68, 78 (SEQ ID NO. 223) krqssgimktegtlencetkcqtplgainttlpfhnvh 1957,59, 83 (SEQ ID NO. 224) kgsnyp(v/i)ak(g/r)synntsgeqmliiwq(v/i)h 1957,58, 59, 61, 83, 91, 95 (SEQ ID NO. 225)httlgqsracavsgnpsffmmvwltekgsnypvak 1957 (SEQ ID NO. 226) khfekvk 1957,59, 65 (SEQ ID NO. 227) kiskrgssgimktegrlencetkcqtplgainttlpfh 1957, 59,65, 91 (SEQ ID NO. 228) krgssgimktegtlencetkcqtplgainttlpfh 1957, 59,65, 91 (SEQ ID NO. 229) ktegtlencetkcqtplgainttlpfh 1957, 59, 65, 91(SEQ ID NO. 230) kiskrgssgimktegtlencetkcqtplgainttlpfh 1957, 59, 65, 91(SEQ ID NO. 231) ktegtlencetkcqtplgainttlpfhn(v/i)h 1957, 59, 65, 91(SEQ ID NO. 232) kiskrgssgimktegtlencetkcqtplgainttlpfh 1957, 59, 65, 91(SEQ ID NO. 233) k(e/g)snypvakgsynntsgeqmliiwgvh 1957, 60, 65 (SEQ IDNO. 234) hpltigccpkyvksek 1957, 60, 65 (SEQ ID NO. 235) kcqtplgaikttlpfh1957, 65 (SEQ ID NO. 236) hhsndqgsgyaadkestqka(f/i)dgitnkvnsviek- 1961,65, 68, 83, 84        -mntqfeavgklf(n/s)nleklenlnkk (SEQ ID NO. 237)hsndqgsgyaadkestqka(f/i)dgitnkvnsviek- 1961, 65, 68, 83, 84       -mntqfeavgklf(n/s)nleklenlnkk (SEQ ID NO. 238)hsndqgsgyaadkestqka(f/i)dgitnk 1961, 65, 68, 83, 84 (SEQ ID NO. 239)hdsnvrnlydkvrmqlrdnak 1964, 68, 76, 84, 91 (SEQ ID NO. 240)hkcddecmnsvkngtydypklnrneikgvk 1964, 65, 68, 76, 83, 84, 91 (SEQ ID NO.241) hkcddecmnsvkngtydypklnrneik 1964, 65, 68, 76, 83, 84, 91 (SEQ IDNO. 242) hkcddecmnsvkngtydypk 1964, 65, 68, 76, 83, 84, 91 (SEQ ID NO.243) hkcddecmnsvk 1964, 65, 68, 76, 83, 84, 91 (SEQ ID NO. 244)kgsnypvakgsynntngeqiliiwgvh 1976, 78 (SEQ ID NO. 245)hsndqgsgyaadkestqkavdgitnkvnsviekmntqfeavgk 1976, 91 (SEQ ID NO. 246)krgssgimktegtlencetkcqtplgainttlpfh 1976, 78, 83, 84 (SEQ ID NO. 247)hpltigecpkyvksek 1976 (SEQ ID NO. 248) hakdilekthngklck 1976 (SEQ ID NO.249) 1. Influenza H2N2 was responsible for the human pandemic (globaldistribution) of 1957. 2. Abbreviation for years: eg. “58” = 1958. 3.The first year that a given Replikin appears is indicated at thebeginning of the series of years in which that Replikin has been foundin this work. 4. Overlapping Replikin sequences are listed separately.5. Increase in number of new Replikin structures occurs in years ofepidemics (underlined): eg. 1957 and 1965 and correlates with increasedtotal Replikin concentration (number of Replikins per 100 amino acidresidues). See FIG. 8.

TABLE 6 H3N2 Replikin Sequences present in H3N2 hemagglutinins ofInfluenza viruses in each year for which amino acid sequences wereavailable (1968-2000) Influenza H3N2 Replikins Year Detected inInfluenza H3N2 strain Influenza Replikins (Peak in FIG. 8: P3 E3 E4)hdvyrdealnnrfqikgvelksgyk 1968, 72, 75 96, 97, 98 (SEQ ID NO. 250)htidltdsemnklfertrk 1968 (SEQ ID NO. 251) kfhqiek 1968, 72, 75, 77 96,97, 98 (SEQ ID NO. 252) ktnekfh(g/q)iek 1968 86 98 (SEQ ID NO. 253)klnr(v/l)iektnekfh 1968, 72, 75, 77 97, 98 (SEQ ID NO. 254)hqiekefsevegriqdlekyvedtk 1968, 72, 98 (SEQ ID NO. 255) kicnnphk 1975(SEQ ID NO. 256) klnrvikktnekfh 1975 (SEQ ID NO. 257)hd(l,v)yrdealnnrfqik(g/q)ve(r/k)s(q/g)yk 1975, 76, 77, 86 (SEQ ID NO.258) hqiekefsevegriqdlekyvedtk 1975 (SEQ ID NO. 259)kyvedtkidlwsynaellvalenqh 1975 (SEQ ID NO. 260)kyvkqnslklatgmrnvpekqtrglfgaiagfiengwegmidgwygfrh 1975 (SEQ ID NO. 261)kefsevegriqdlekyvedtkidlwsynaellvalenqh 1975 2000 (SEQ ID NO. 262)hqn(s/e)(e/q)g(t/s)g(q/y)aad(l/q)k- 1975 2000-stq(a/n)a(i/l)d(q/g)l(n/t)(g/n)k(l/v)n(r/s)vi(e/c)k (SEQ ID NO. 263)hcd(g/q)f(q,r)nekwdlf(v,/i)er(s/t)k 1975, 76, 77, 78, 80, 81, 82, (SEQID NO. 264) 83, 84, 85, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98htidltdsemnkklfertrk 1977, (SEQ ID NO. 265) ksgstypvlkvtmpnndnfdklyiwgvh1977 (SEQ ID NO. 266) klnwltksgntypvlnvtmpnndnfdklviwgvh 1982 (SEQ IDNO. 267) htidltdsemnklfektrk 1986 (SEQ ID NO. 268) klnrliektnekfhqtek1987 (SEQ ID NO. 269) htgkssvmrsdapidfcnsecitpnqsipndkpfqnvnkitygacpk1994 (SEQ ID NO. 270) htgkssvmrsdapidfcnsecitpnqsipndkpfqnvnk 1994 (SEQID NO. 271) hpstdsdqtslyvrasgrvtvstkrsqqtvipk 1994 (SEQ ID NO. 272)kyvedtkidlwsynaellvalenqh 1997, 98 (SEQ ID NO. 273)klfertrkqlrenaedmgngcfkiyh 1998 (SEQ ID NO. 274) krrsiksffsrlnwlh 1998(SEQ ID NO. 275) hpvtigecpky(v/r)kstk 2000 (SEQ ID NO. 276)kgnsypklsklsksyiinkkkevlviwgih 2000 (SEQ ID NO. 277)klsklsks(v/y)iinkkkevlviwgih 2000 (SEQ ID NO. 278)klsks(v/y)iinkkkevlviwgih 2000 (SEQ ID NO. 279) 1. Influenza H3N2 wasresponsible for the human pandemic (global distribution) of 1968. 2.Abbreviation for years: eg. “77” = 1977. 3. The first year that a givenReplikin appears is indicated at the beginning of the series of years inwhich that Replikin has been found. 4. Overlapping Replikin sequencesare listed separately. 5. Increase in number of new Replikin structuresoccurs in years of epidemics (underlined) : eg. 1975 and correlates withincreased total Replikin concentration (number of Replikins per 100amino acid residues). See FIG. 8.

Both the concentration and type, i.e., composition of Replikinsobserved, were found to relate to the occurrence of influenza pandemicsand epidemics. The concentration of Replikins in influenza viruses wasexamined by visually scanning the hemagglutinin amino acid sequencespublished in the National Library of Medicine “PubMed” data base forinfluenza strains isolated world wide from human and animal reservoirsyear by year over the past century, i.e., 1900 to 2001. These Replikinconcentrations (number of Replikins per 100 amino acids, mean+/−SD) werethen plotted for each strain.

The concentration of Replikins was found to directly relate to theoccurrence of influenza pandemics and epidemics. The concentration ofReplikins found in influenza B hemagglutinin and influenza A strain,H1N1, is shown in FIG. 7, and the concentration of Replikins found inthe two other common influenza virus A strains, H2N2 and H3N2 is shownin FIG. 8 (H2N2, H3N2). The data in FIG. 8 also demonstrate an emergingnew strain of influenza virus as defined by its constituent Replikins(H3N2(R)).

Each influenza A strain has been responsible for one pandemic: in 1918,1957, and 1968, respectively. The data in FIGS. 7 and 8 show that atleast one Replikin per 100 amino acids is present in each of theinfluenza hemagglutinin proteins of all isolates of the four commoninfluenza viruses examined, suggesting a function for Replikins in themaintenance of survival levels of replication. In the 1990s, during thedecline of the H3N2 strain, there were no Replikins in many isolates ofH3N2, but a high concentration of new Replikins appeared in H3N2isolates, which define the emergence of the H3N2(R) strain.

Several properties of Replikin concentration are seen in FIG. 7 and FIG.8 to be common to all four influenza virus strains. First, theconcentration is cyclic over the years, with a single cycle of rise andfall occurring over a period of two to thirty years. This rise and fallis consistent with the known waxing and waning of individual influenzavirus strain predominance by hemagglutinin and neuraminidaseclassification. Second, peak Replikin concentrations of each influenzavirus strain previously shown to be responsible for a pandemic wereobserved to relate specifically and individually to each of the threeyears of the pandemics. For example, for the pandemic of 1918, where theinfluenza virus strain, H1N1, was shown to be responsible, a peakconcentration of the Replikins in H1N1 independently occurred (P1); forthe pandemic of 1957, where H2N2 emerged and was shown to beresponsible, a peak concentration of the Replikins in H2N2 occurred(P2); and for the pandemic of 1968, where H3N2 emerged and was shown tobe the cause of the pandemic, a peak concentration of the Replikins inH3N2 occurred (P3). Third, in the years immediately following each ofthe above three pandemics, the specific Replikin concentration decreasedmarkedly, perhaps reflecting the broadly distributed immunity generatedin each case. Thus, this post-pandemic decline is specific for H1N1immediately following the pandemic (P1) for which it was responsible,and is not a general property of all strains at the time. An increase ofReplikin concentration in influenza B repeatedly occurred simultaneouslywith the decrease in Replikin concentration in H1N1, e.g., EB1 in 1951and EB2 in 1976, both associated with influenza B epidemics having thehighest mortality. (Stuart-Harris, et al., Edward Arnold Ltd. (1985).Fourth, a secondary peak concentration, which exceeded the primary peakincrease in concentration, occurred 15 years after each of the threepandemics, and this secondary peak was accompanied by an epidemic: 15years after the 1918 pandemic in an H1N1 ‘epidemic’ year (E1); eightyears after the 1957 pandemic in an H2N2 ‘epidemic’ year (E2); andoccurred seven years after the 1968 pandemic in an H3N2 ‘epidemic’ year(E3). These secondary peak concentrations of specific Replikins mayreflect recovery of the strain. Fifth, peaks of each strain's specificReplikin concentration frequently appear to be associated with declinesin Replikin concentration of one or both other strains, suggestingcompetition between strains for host sites. Sixth, there is an apparentoverall tendency for the Replikin concentration of each strain todecline over a period of 35 years (H2N2) to 60 years (influenza B). Thisdecline cannot be ascribed to the influence of vaccines because it wasevident in the case of influenza B from 1901 to 1964, prior to commonuse of influenza vaccines. In the case of influenza B, Replikin recoveryfrom the decline is seen to occur after 1965, but Replikin concentrationdeclined again between 1997 and 2000 (FIG. 7). This correlates with thelow occurrence of influenza B in recent case isolates. H1N1 Replikinconcentration peaked in 1978-1979 (FIG. 7) together with thereappearance and prevalence of the H1N1 strain, and then peaked in 1996coincident with an H1N1 epidemic. (FIG. 7). H1N1 Replikin concentrationalso declined between 1997 and 2000, and the presence of H1N1 strainsdecreased in isolates obtained during these years. For H2N2 Replikins,recovery from a 35 year decline has not occurred (FIG. 8), and thiscorrelates with the absence of H2N2 from recent isolates. For H3N2, theReplikin concentration of many isolates fell to zero during the periodfrom 1996 to 2000, but other H3N2 isolates showed a significant, sharpincrease in Replikin concentration. This indicates the emergence of asubstrain of H3N2, which is designated herein as H3N2(R).

FIGS. 7 and 8 demonstrate that frequently, a one to three year stepwiseincrease is observed before Replikin concentration reaches a peak. Thisstepwise increase proceeds the occurrence of an epidemic, which occursconcurrently with the Replikin peak. Thus, the stepwise increase inconcentration of a particular strain is a signal that particular strainis the most likely candidate to cause an epidemic or pandemic.

Currently, Replikin concentration in the H3N2(R) strain of influenzavirus is increasing (FIG. 8, 1997 to 2000). Three similar previous peakincreases in H3N2 Replikin concentration are seen to have occurred inthe H3N2-based pandemic of 1968 (FIG. 8), when the strain first emerged,and in the H3N2-based epidemics of 1972 and 1975 (FIG. 8). Each of thesepandemic and epidemics was associated with excess mortality. (Ailing, etal., Am J. Epidemiol., 113(1):30-43 (1981). The rapid ascent inconcentration of the H3N2(R) subspecies of the H3N2 Replikins in1997-2000, therefore, statistically represents an early warning of anapproaching severe epidemic or pandemic. An H3N2 epidemic occurred inRussia in 2000 (FIG. 8, E4); and the CDC report of December 2001 statesthat currently, H3N2 is the most frequently isolated strain of influenzavirus world wide. (Morbidity and Mortality Weekly Reports (MMWR), Centerfor Disease Control; 50(48):1084-68 (Dec. 7, 2001).

In each case of influenza virus pandemic or epidemic new Replikinsemerge. There has been no observation of two of the same Replikins in agiven hemagglutinin in a given isolate. To what degree the emergence ofa new Replikin represents mutations versus transfer from another animalor avian pool is unknown. In some cases, each year one or more of theoriginal Replikin structures is conserved, while at the same time, newReplikins emerge. For example, in influenza virus B hemagglutinin, fiveReplikins were constantly conserved between 1919 and 2001, whereas 26Replikins came and went during the same period (some recurred afterseveral years absence). The disappearance and re-emergence years laterof a particular Replikin structure suggests that the Replikins returnfrom another virus host pool rather than through de novo mutation.

In the case of II1N1 Replikins, the two Replikins present in the P1 peakassociated with the 1918 pandemic were not present in the recovery E1peak of 1933, which contains 12 new Replikins. Constantly conservedReplikins, therefore, are the best choice for vaccines, either alone orin combination. However, even recently appearing Replikins accompanyingone year's increase in concentration frequently persist and increasefurther for an additional one or more years, culminating in aconcentration peak and an epidemic, thus providing both an early warningand time to vaccinate with synthetic Replikins (see for example, H1N1 inthe early 1990's, FIG. 7).

The data in FIGS. 7 and 8 demonstrate a direct relationship between thepresence and concentration of a particular Replikin in influenza proteinsequences and the occurrence of pandemics and epidemics of influenza.Thus, analysis of the influenza virus hemagglutinin protein sequence forthe presence and concentration of Replikins provides a predictor ofinfluenza pandemics and/or epidemics, as well as a target for influenzavaccine formulation.

Composition of Replikins in Strains of Influenza Virus B: Of a total of26 Replikins identified in this strain (Table 3), the following tenReplikins are present in every influenza B isolate examined from1902-2001. Overlapping Replikin sequences are listed separately. Lysinesand histidines are in bold type to demonstrate homology consistent withthe “3-point recognition.”

kshfanlk (SEQ ID NO. 91) kshfanlkgtk (SEQ ID NO. 92) kshfanlkgtktrgklcpk(SEQ ID NO. 93) hekygglnk (SEQ ID NO. 94) hekygglnksk (SEQ ID NO. 95)hekygglnkskpyytgehak (SEQ ID NO. 96) hakaigncpiwvk (SEQ ID NO. 97)hakaigncpiwvvkktplklangtk (SEQ ID NO. 98) hakaigncpiwvktplklangtkyrppak(SEQ ID NO. 99) hakaigncpiwvktplklangtkyrppakllk (SEQ ID NO. 100)

Tables 3 and 4 indicate that there appears to be much greater stabilityof the Replikin structures in influenza B hemagglutinins compared withH1N1 Replikins. Influenza B has not been responsible for any pandemic,and it appears not to have an animal or avian reservoirs. (Stuart-Harriset al., Edward Arnold Ltd., London (1985)).

Influenza H1N1 Replikins: Only one Replikin“hp(v/i)tigecpkyv(r/k)(s/t)(t/a)k” is present in every H1N1 isolate forwhich sequences are available from 1918, when the strain first appearedand caused the pandemic of that year, through 2000 (Table 4) (“(v/i)”indicates that the amino acid v or i is present in the same position indifferent years.) Although H1N1 contains only one persistent Replikin,H1N1 appears to be more prolific than influenza B. There are 95different Replikin structures in 82 years on H1N1 versus only 31different Replikins in 100 years of influenza B isolates (Table 4). Anincrease in the number of new Replikin structures occurs in years ofepidemics (Tables 3, 4, 5 and 6) and correlates with increased totalReplikin concentration (FIGS. 7 and 8).

Influenza H2N2 Replikins: Influenza H2N2 was responsible for the humanpandemic of 1957. Three of the 20 Replikins identified in that strainfor 1957 were conserved in each of the H2N2 isolates available forexamination on PubMed until 1995 (Table 5).

(SEQ ID NO. 219) ha(k/q/m)(d/n)ilekthngk (SEQ ID NO. 220)ha(k/q/m)(d/n)ilekthngklc(k/r) (SEQ ID No. 225)kgsnyp(v/i)ak(g/r)synntsgeqmliiwq(v/i)h

However, in contrast to H1N1, only 13 additional Replikins have beenfound in H2N2 beginning in 1961. This paucity of appearance of newReplikins correlates with the decline in the concentration of the H2N2Replikins and the appearance of H2N2 in isolates over the years (FIG.8).

Influenza H3N2 Replikins: Influenza H3N2 was responsible for the humanpandemic of 1968. Five Replikins which appeared in 1968 disappearedafter 1977, but reappeared in the 1990s (Table 6). The only Replikinstructure which persisted for 22 years washcd(g/q)f(q/r)nekwdlf(v/i)er(s/t)k, which appeared first in 1977 andpersisted through 1998. The emergence of twelve new H3N2 Replikins inthe mid 1990s (Table 6) correlates with the increase in Replikinconcentration at the same time (FIG. 8), and with the prevalence of theH3N2 strain in recent isolates together with the concurrentdisappearance of all Replikins from some of these isolates (FIG. 8),this suggests the emergence of the new substrain H3N2(R).

FIGS. 1 and 2 show that influenza epidemics and pandemics correlate withthe increased concentration of Replikins in influenza virus, which isdue to the reappearance of at least one Replikin from one to 59 yearsafter its disappearance. Also, in the A strain only, there is anemergence of new strain-specific Replikin compositions (Tables 4-6).Increase in Replikin concentration by repetition of individual Replikinswithin a single protein appears not to occur in influenza virus, but isseen in other organisms.

It has been believed that changes in the activity of different influenzastrains are related to sequence changes in influenza hemagglutinins,which in turn are the products of substitutions effected by one of twopoorly understood processes: i) antigenic drift, thought to be due tothe accumulation of a series of point mutations in the hemagglutininmolecule, or ii) antigenic shift, in which the changes are so great thatgenetic reassortment is postulated to occur between the viruses of humanand non-human hosts. First, the present data suggests that the change inactivity of different influenza strains, rather than being related tonon-specific sequence changes, are based upon, or relate to theincreased concentration of strain-specific Replikins and strain-specificincreases in the replication associated with epidemics. In addition, thedata were examined for a possible insight into which sequence changesare due to “drift” or “shift”, and which are due to conservation,storage in reservoirs, and reappearance. The data show that theepidemic-related increase in Replikin concentration is not due to theduplication of existing Replikins per hemagglutinin, but is due to thereappearance of at least one Replikin composition from 1 to up to 59years after its disappearance, plus in the A strains only, the emergenceof new strain-specific Replikin compositions (Tables 3-6). Thus theincrease in Replikin concentration in the influenza B epidemics of 1951and 1977 are not associated with the emergence of new Replikincompositions in the year of the epidemic but only with the reappearanceof Replikin compositions which had appeared in previous years thendisappeared (Table 3). In contrast, for the A strains, in addition tothe reappearance of previously disappeared virus Replikins, newcompositions appear (e.g. in H1N1 in the year of the epidemic of 1996,in addition to the reappearance of 6 earlier Replikins, 10 newcompositions emerged). Since the A strains only, not influenza B, haveaccess to non-human animal and avian reservoirs, totally newcompositions probably derive from non-human host reservoirs rather thanfrom mutations of existing human Replikins which appear to bear noresemblance to the new compositions other than the basic requirements of“3-point recognition” (Tables 2-5). The more prolific nature of H1N1compared with B, and the fact that pandemics have been produced by thethree A strains only, but not by the B strain, both may also be afunction of the ability of the human A strains to receive new Replikincompositions from non-human viral reservoirs.

Some Replikins have appeared in only one year, disappeared, and notreappeared to date (Tables 3-6). Other Replikins disappear from one toup to 81 years, when the identical Replikin sequence reappears. KeyReplikin ‘k’ and ‘h’ amino acids, and the spaces between them, areconserved during the constant presence of particular Replikins over manyyears, as shown in Tables 23-6 for the following strain-specificReplikins: ten of influenza B, the single Replikin of H1N1, and thesingle Replikin of H2N3, as well as for the reappearance of identicalReplikins after an absence. Despite the marked replacement orsubstitution activity of other amino acids both inside the Replikinstructure and outside it in the rest of the hemagglutinin sequences,influenza Replikin histidine (h) appears never to be, and lysine (k) israrely replaced. Examples of this conservation are seen in the H1N1Replikin “hp(v/i)tigecpkyv(r/k)(s/t)(t/a)k,” (SEQ ID NO. 122) constantbetween 1918 and 2000, in the H3N2 Replikin“hcd(g/q)f(q,r)nekwdlf(v/i)er(s/t)k” (SEQ ID NO. 264) constant between1975 and 1998 and in the H3N2 Replikin“hqn(s/e)(e/q)g(t/s)g(q/y)aad(1/q)kstq(a/n)a(i/l)d(q/g)l(n/t)(g/n)k(1/v)n(r/s)vi(e/c)k” (SEQ ID NO. 263) which first appeared in 1975, disappeared for25 years, and then reappeared in 2000. While many amino acids weresubstituted, the basic Replikin structure of 2 Lysines, 6 to 10 residuesapart, one histidine, a minimum of 6% lysine in not more thanapproximately 50 amino acids, was conserved.

Totally random substitution would not permit the persistence of theseH1N1 and H3N2 Replikins, nor from 1902 to 2001 in influenza B thepersistence of 10 Replikin structures, nor the reappearance in 1993 of a1919 18 mer Replikin after an absence of 74 years. Rather than a randomtype of substitution, the constancy suggests an orderly controlledprocess, or in the least, protection of the key Replikin residues sothat they are fixed or bound in some way: lysines, perhaps bound tonucleic acids, and histidines, perhaps bound to respiratory redoxenzymes. The mechanisms which control this conservation are at presentunknown.

Conservation of Replikin Structures

Whether Replikin structures are conserved or are subject to extensivenatural mutation was examined by scanning the protein sequences ofvarious isolates of foot and mouth disease virus (FMDV), where mutationsin proteins of these viruses have been well documented worldwide fordecades. Protein sequences of FMDV isolates were visually examined forthe presence of both the entire Replikin and each of the componentReplikin amino acid residues observed in a particular Replikin.

Rather than being subject to extensive substitution over time as occursin neighboring amino acids, the amino acids which comprise the Replikinstructure are substituted little or not at all, that is the Replikinstructure is conserved.

For example, in the protein VP1 of FMDV type O, the Replikin (SEQ IDNO.: 3) “hkqkivapvk” was found to be conserved in 78% of the 236isolates reported in PubMed, and each amino acid was found to beconserved in individual isolates as follows: his, 95.6%; lys, 91.8%; gin92.3%; lys, 84.1%; ile, 90.7%; val, 91.8%; ala, 97.3%; pro, 96.2%; ala,75.4%; and lys, 88.4%. The high rate of conservation suggests structuraland functional stability of the Replikin structure and provides constanttargets for treatment.

Similarly, sequence conservation was found in different isolates of HIVfor its Replikins, such as (SEQ ID NO.: 5) “kcfncgkegh” or (SEQ ID NO.:6) “kvylawvpahk” in HIV Type 1 and (SEQ ID NO.: 7) “kcwncgkegh” in HIVType 2 (Table 2). Further examples of sequence conservation were foundin the HIV tat proteins, such as (SEQ ID NO.: 698)“hclvckqkkglgisygrkk,” wherein the key lysine and histidine amino acidsare conserved (See Table 7).

Similarly, sequence conservation was observed in plants, for example inwheat, such as in wheat ubiguitin activating enzyme E (SEQ ID NOs.601-603). The Replikins in wheat even provided a reliable target forstimulation of plant growth as described within. Other examples ofconservation are seen in the constant presence of malignin in successivegenerations, over ten years of tissue culture of glioma cells, and bythe constancy of affinity of the glioma Replikin for antimaligninantibody isolated by immunoadsorption from 8,090 human sera from theU.S., U.K., Europe and Asia (e.g., FIG. 5 and U.S. Pat. No. 6,242,578B1).

Similarly, conservation was observed in trans-activator (Tat) proteinsin isolates of HIV. Tat (trans-activator) proteins are early RNA bindingproteins regulating lentiviral transcription. These proteins arenecessary components in the life cycle of all known lentiviruses, suchas the human immunodeficiency viruses (HIV). Tat is a transcriptionalregulator protein that acts by binding to the trans-activating responsesequence (TAR) RNA element and activates transcription Initiation and/orelongation from the LTR promoter. HIV cannot replicate without tat, butthe chemical basis of this has been unknown. In the HIV tat proteinsequence from 89 to 102 residues, we have found a Replikin that isassociated with rapid replication in other organisms. The amino acidsequence of this Replikin is “hclvcfqkkglgisygrkk.” In fact, we foundthat this Replikin is present in every HIV tat protein. Some tat aminoacids are substituted frequently, as shown in Table 8, by alternateamino acids (in small size fonts lined up below the most frequent aminoacid (Table 7), the percentage of conservation for the predominantReplikin “hclvcfqkkglgisygrkk”). These substitutions have appeared formost of the individual amino acids. However, the key lysine andhistidine amino acids within the Replikin sequence, which define theReplikin structure, are conserved 100% in the sequence; whilesubstitutions are common elsewhere in other amino acids, both within andoutside the Replikin, none occurs on these key histidine amino acids.

As shown in Table 7, it is not the case that lysines are not substitutedin the tat protein amino acid sequence. From the left side of the table,the very first lysine in the immediate neighboring sequence, but outsidethe Replikin sequence, and the second lysine (k) in the sequence insidethe Replikin, but “extra” in that it is not essential for the Replikinformation, are both substituted frequently. However, the 3rd, 4th and5th lysines, and the one histidine, in parentheses, which together setup the Replikin structure, are never substituted. Thus, these key aminoacid sequences are 100% conserved. As observed in the case of theinfluenza virus Replikins, random substitution would not permit thisselective substitution and selective non-substitution to occur due tochance.

TABLE 7 % Replikin CONSERVATION of each constituent amino acid in thefirst 117 different isolates of HIV tat protein as reported in PubMed:38 100 57 86 100 100 66 76 100 99 57 49 100 94 100 97 98 85 97 99 100100 100% Neighboring-Amino acids [tat Replikin] k (c) s y [(h) (c) l v(c) f q k (k) g (I) g i s y g (r) (k) (k)] below are the amino acidsubstitutions observed for each amino acid above: h c f q i l h t a a ly h q r w p l l i h q v y s s l m r s i s m s s r n v a f p q

The conservation of the Replikin structure suggests that the Replikinstructure has a specific survival function for the HIV virus which mustbe preserved and conserved, and cannot be sacrificed to the virus‘defense’ maneuver of amino acid substitution crested to avoid antibodyand other ‘attack.’ These ‘defense’ functions, although also essential,cannot ‘compete’ with the virus survival function of HIV replication.

Further conservation was observed in different isolates of HIV for itsReplikins such as “kcfncgkegh” (SEQ ID NO. 5) or “kvylawvpahk” (SEQ IDNO. 6) in HIV Type 1 and “kcwncgkegh” (SEQ ID NO. 7) in HIV Type 2.

The high rate of conservation observed in FMVD and HIV Replikinssuggests that conservation also observed in the Replikins of influenzaReplikins is a general property of viral Replikins. This conservationmakes them a constant and reliable tarted for either destruction, forexample by using specific Replikins such as for influenza, FMVD or HIVvaccines as illustrated for the glioma Replikin, or stimulation.

Similarly, as provided in examples found in viruses including influenzaviruses, FMDV, and HIV, where high rates of conservation in Replikinssuggest that conservation is a general property of viral Replikins andthus making Replikins a constant and reliable target for destruction orstimulation, conservation of Replikin structures occurs in plants. Forexample, in wheat plants, Replikins are conserved and provide a reliabletarget for stimulation. Examples of conserved Replikins in wheat plantsubiquitin activating enzyme E include:

E3 hkdrltkkvvdiarevakvdvpeyrrh (SEQ ID NO. 601) E2hkerldrkvvdvarevakvevpsyrrh (SEQ ID NO. 602) E1hkerldrkvvdvarevakmevpsyrrh (SEQ ID NO. 603)   *  *         *  **  *

Similarly to conservation found in the HIV tat protein, the Replikin inthe wheat ubiquitin activating enzyme E is conserved. As with the HIVtat protein, substitutions of amino acids (designated with an ‘*’)adjacent to the Replikin variant forms in wheat ubiquitin activatingenzyme E are common. The key k and h amino acids that form the Replikinstructure, however, do not vary whereas the ‘unessential’ k that is only5 amino acids (from the first k on the left) is substituted.

Anti-Replikin Antibodies

An anti-Replikin antibody is an antibody against a Replikin. Data onanti-Replikin antibodies also support Replikin class unity. Ananti-Replikin antibody response has been quantified by immunoadsorptionof serum antimalignin antibody to immobilized malignin (see Methods inU.S. Pat. No. 5,866,690). The abundant production of antimaligninantibody by administration to rabbits of the synthetic version of the16-mer peptide whose sequence was derived from malignin, absentcarbohydrate or other groups, has established rigorously that thispeptide alone is an epitope, that is, provides a sufficient basis forthis immune response (FIG. 3). The 16-mer peptide produced both IgM andIgG forms of the antibody. Antimalignin antibody was found to beincreased in concentration in serum in 37% of 79 cases in the U.S. andAsia of hepatitis B and C, early, in the first five years of infection,long before the usual observance of liver cancer, which develops aboutfifteen to twenty-five years after infection. Relevant to bothinfectious hepatitis and HIV infections, transformed cells may be oneform of safe haven for the virus: prolonging cell life and avoidingvirus eviction, so that the virus remains inaccessible to anti-viraltreatment.

Because administration of Replikins stimulates the immune system toproduce antibodies having a cytotoxic effect, peptide vaccines based onthe particular influenza virus Replikin or group of Replikins observedto be most concentrated over a given time period provide protectionagainst the particular strain of influenza most likely to cause anoutbreak in a given influenza season, e.g., an emerging strain orre-emerging strain For example, analysis of the influenza virushemagglutinin amino acid sequence on a yearly or bi-yearly basis,provides data which are useful in formulating a specifically targetedinfluenza vaccine for that year. It is understood that such analysis maybe conducted on a region-by-region basis or at any desired time period,so that strains emerging in different areas throughout the world can bedetected and specifically targeted vaccines for each region can beformulated.

Influenza

Currently, vaccine formulations are changed twice yearly atinternational WHO and CDC meetings. Vaccine formulations are based onserological evidence of the most current preponderance of influenzavirus strain in a given region of the world. However, prior to thepresent invention there has been no correlation of influenza virusstrain specific amino acid sequence changes with occurrence of influenzaepidemics or pandemics.

The observations of specific Replikins and their concentration ininfluenza virus proteins provides the first specific quantitative earlychemical correlates of influenza pandemics and epidemics and providesfor production and timely administration of influenza vaccines tailoredspecifically to treat the prevalent emerging or re-emerging strain ofinfluenza virus in a particular region of the world. By analyzing theprotein sequences of isolates of strains of influenza virus, such as thehemagglutinin protein sequence, for the presence, concentration and/orconservation of Replikins, influenza virus pandemics and epidemics canbe predicted. Furthermore, the severity of such outbreaks of influenzacan be significantly lessened by administering an influenza peptidevaccine based on the Replikin sequences found to be most abundant orshown to be on the rise in virus isolates over a given time period, suchas about one to about three years.

An influenza peptide vaccine of the invention may include a singleReplikin peptide sequence or may include a plurality of Replikinsequences observed in influenza virus strains. Preferably, the peptidevaccine is based on Replikin sequence(s) shown to be increasing inconcentration over a given time period and conserved for at least thatperiod of time. However, a vaccine may include a conserved Replikinpeptide(s) in combination with a new Replikin(s) peptide or may be basedon new Replikin peptide sequences. The Replikin peptides can besynthesized by any method, including chemical synthesis or recombinantgene technology, and may include non-Replikin sequences, althoughvaccines based on peptides containing only Replikin sequences arepreferred. Preferably, vaccine compositions of the invention alsocontain a pharmaceutically acceptable carrier and/or adjuvant.

The influenza vaccines of the present invention can be administeredalone or in combination with antiviral drugs, such as gancyclovir;interferon; interleukin; M2 inhibitors, such as, amantadine,rimantadine; neuraminidase inhibitors, such as zanamivir andoseltamivir; and the like, as well as with combinations of antiviraldrugs.

Replikin Decoys in Malaria

Analysis of the primary structure of a Plasmodium falciparum malariaantigen located at the merozoite surface and/or within theparasitophorous vacuole revealed that this organism, like influenzavirus, also contains numerous Replikins (Table 8). However, there areseveral differences between the observation of Replikins in Plasmodiumfalciparum and influenza virus isolates. For example, Plasmodiumfalciparum contains several partial Replikins, referred to herein as“Replikin decoys.” These decoy structures contain an abundance of lysineresidues, but lack the histidine required of Replikin structures.Specifically, these decoys contain many lysines 6 to 10 residues apartin overlapping fashion, similar to the true malaria recognins butwithout histidine residues. It is believed that the decoy structuremaximizes the chances that an anti-malarial antibody or other agent willbind to the relatively less important structure containing the lysines,i.e., the Replikin decoys, rather than binding to histidine, which ispresent in Replikin structure, such as Replikins in respiratory enzymes,which could result in destruction of the trypanosome. For example, anincoming antibody, with specificity for Replikin structures, mightattach to the Replikin decoy structure, leaving the true Replikinstructure remains untouched.

Therefore, anti-Replikin treatment of malaria requires two phases (dualtreatment): i) preliminary treatment with proteolytic enzymes thatcleave the Replikin decoys, permitting ‘safe passage’ of the specificanti-Replikin treatment; and ii) attacking malaria Replikins either withspecific antibodies or by cellular immunity engendered by syntheticmalaria Replikin vaccines or by organic means targeting the malariaReplikins.

Repetition and Overlapping of Replikin Structures

Another difference seen in Plasmodium falciparum is a frequentrepetition of individual Replikin structures within a single protein,which was not observed with influenza virus. Repetition may occur by (a)sharing of lysine residues between Replikins, and (b) by repetition of aportion of a Replikin sequence within another Replikin sequence.

A third significant difference between Replikin structures observed ininfluenza virus isolates and Plasmodium falciparum is a markedoverlapping of Replikin structures throughout malarial proteins, e.g.,there are nine overlapping Replikins in the 39 amino acid sequence ofSEQ ID NO. 380 (Replikin concentration=23.1/100 amino acids); and 15overlapping Replikins in the 41 amino acids of SEQ ID NO. 454 (Replikinconcentration=36.6/100 amino acids). Both of these overlapping Replikinstructures occur in blood stage trophozoites and schizonts. In contrast,influenza virus Replikins are more scattered throughout the protein andthe maximum Replikin concentration is about 7.5/100 amino acids (FIG.7); and tomato leaf curl gemini virus, which was also observed to haveoverlapping Replikins has only about 3.1/100 amino acids.

This mechanism of lysinc multiples is also seen in the Replikins ofcancer proteins such as in gastric cancer transforming protein,ktkkgnrvsptmkvth (SEQ ID NO. 88), and in transforming protein P21B(K-RAS 2B) of lung, khkekmskdgkkkkkks (SEQ ID NO. 89).

The relationship of higher Replikin concentration to rapid replicationis also confirmed by analysis of HIV isolates. It was found that theslow-growing low titer strain of HIV (NS, “Bru,” which is prevalent inearly stage HIV infection) has a Replikin concentration of 1.1 (+/−1.6)Replikins per 100 amino acids, whereas the rapidly-growing high titerstrain of HIV (SI, “Lai”, which is prevalent in late stage HIVinfection) has a Replikin concentration of 6.8 (+/−2.7) Replikins per100 amino acid residues.

The high concentration of overlapping Replikins in malaria, influenzavirus and cancer cells is consistent with the legendary high and rapidreplicating ability of malaria organisms. The multitude of overlappingReplikins in malaria also provides an opportunity for the organism toflood and confuse the immune system of its host and thereby maximize thechance that the wrong antibody will be made and perpetuated, leaving keymalaria antigens unharmed.

As in the case of influenza virus, for example, peptide vaccines basedon the Replikin structure(s) found in the malaria organism can providean effective means of preventing and/or treating malaria. Vaccinationagainst malaria can be achieved by administering a compositioncontaining one or a mixture of Replikin structures observed inPlasmodium falciparum. Furthermore, antibodies to malaria Replikins canbe generated and administered for passive immunity or malaria detection

Table 8 provides a list of several Plasmodium falciparum Replikinsequences. It should be noted that this list is not meant to becomplete. Different isolates of the organism may contain other Replikinstructures.

TABLE 8 Malaria Replikins a) Primary structure of a Plasmodiumfalciparum malaria antigen located at the merozoite surface and withinthe parasitophorous vacuole a) i) DECOYS: (C-Terminal)keeeekekekekekeekekeekekeekekekeekekekeekeeekk, (SEQ ID NO. 280) orkeeeekekekekekeekekeekekeekekekeekekekeekeeekkek, (SEQ ID NO. 281) orkeeeekekekekekeekekeekekekeekekeekekeekeekeeekk, (SEQ ID NO. 282) orkeeeekekek (SEQ ID NO. 283) ii) ReplikinS: Hkklikalkkniesiqnkk (SEQ IDNO. 284) hkklikalkkniesiqnkm (SEQ ID NO. 285) hkklikalkk (SEQ ID NO.286) hkklikalk (SEQ ID NO. 287) katysfvntkkkiislksqghkk (SEQ ID NO. 288)katysfvntkkkiislksqghk (SEQ ID NO. 289) katysfvntkkkiislksqgh (SEQ IDNO. 290) htyvkgkkapsdpqca dikeeckellkek (SEQ ID NO. 291) kiislksqghk(SEQ ID NO. 292) kkkkfeplkngnvsetiklih (SEQ ID NO. 293)kkkfeplkngnvsetiklih (SEQ ID NO. 294) kkfeplkngnvsetiklih (SEQ ID NO.295) kngnvsetiklih (SEQ ID NO. 296) klihlgnkdkk (SEQ ID NO. 297)kvkkigvtlkkfeplkngnvsetiklihlgnkdkkh (SEQ ID NO. 298)hliyknksynplllscvkkmnmlkenvdyiqnqnlfkelmnqkatysfvntkkkiislk (SEQ ID NO.299) hliyknksynplllscvkkmnmlkcnvdyiqnqnlfkelmnqkatysfvntk (SEQ ID NO.300) hliyknksynplllscvkkmnmlkenvdyiqnqnlfkelmnqk (SEQ ID NO. 301)hliyknksynplllscvkkmnmlkenvdyiqknqnlfk (SEQ ID NO. 302)hliyknksynplllscvkkmnmlk (SEQ ID NO. 303)ksannsanngkknnaeemknlvnflqshkklikalkkniesiqnkkh (SEQ ID NO. 304)kknnaeemknlvnflqshkklikalkkniesiqnkkh (SEQ ID NO. 305)knlvnflqshkklikalkkniesiqnkkh (SEQ ID NO. 306) kklikalkkniesiqnkkh (SEQID NO. 307) klikalkkniesiqnkkh (SEQ ID NO. 308) kkniesiqnkkh (SEQ ID NO.309) kniesiqnkkh (SEQ ID NO. 310) knnaeemknlvnflqsh (SEQ ID NO. 311)kklikalkkniesiqnkkqghkk (SEQ ID NO. 312) kknnaeemknlvnflqshk (SEQ ID NO.313) knnaeemknlvnflqsh (SEQ ID NO. 314) klikalkkniesiqnkkqghkk (SEQ IDNO. 315) kvkkigvtlkkfeplkngnvsetiklih (SEQ ID NO. 316) kngnvsetiklih(SEQ ID NO. 317) klihlgnkdkk (SEQ ID NO. 318)ksannsanngkknnaeemknlvnflqsh (SEQ ID NO. 319) kknnaeemknlvnflqsh (SEQ IDNO. 320) kklikalkkniesiqnkkh (SEQ ID NO. 321) kalkkniesiqnkkh (SEQ IDNO. 322) kkniesiqnkkh (SEQ ID NO. 323) kelmnqkatysfvntkkkiislksqgh (SEQID NO. 324) ksqghkk (SEQ ID NO. 325) kkkiislksqgh (SEQ ID NO. 326)kkiislksqgh (SEQ ID NO. 327) kkniesiqnkkh (SEQ ID NO. 328) kniesiqnkkh(SEQ ID NO. 329) htyvkgkkapsdpqcadikeeckellkek (SEQ ID NO. 330)htyvkgkkapsdpqcadikeeckellk (SEQ ID NO. 331) b) “liver stageantigen-3” gene = “LSA-3” Replikins henvlsaalentqseeekkevidvieevk (SEQID NO. 332) kcnvvttilekveettaesvttfsnileeiqentitndtieekleelh (SEQ ID NO.333) hylqqmkekfskek (SEQ ID NO. 334)hylqqmkekfskeknnnvievtnkaekkgnvqvtnktekttk (SEQ ID NO. 335)hylqqmkekfskeknnnvievtnkaekkgnvqvtnktekttkvdknnk (SEQ ID NO. 336)hylqqmkekfskeknnnvievtnkaekkgnvqvtnktekttkvdknnkvpkkrrtqk (SEQ ID NO.337) hylqqmkekfskeknnnvievtnkaekkgnvqvtnktekttkvdknnkvpkkrrtqksk (SEQ IDNO. 338) hvdevmkyvqkidkevdkevskaleskndvtnvlkqnqdffskvknfvkkyk (SEQ IDNO. 339) hvdevmkyvqkidkevdkevskaleskndvtnvlkqnqdffskvknfvkk (SEQ ID NO.340) hvdevmkyvqkidkevdkevskaleskndvtnvlkqnqdffsk (SEQ ID NO. 341)hvdevmkyvqkidkevdkevskaleskndvtnvlk (SEQ ID NO. 342)hvdevmkyvqkidkevdkevskalesk (SEQ ID NO. 343) hvdevmkyvqkidkevdkevsk (SEQID NO. 344) hvdevmkyvqkidkevdk (SEQ ID NO. 345) hvdevmkyvqkidk (SEQ IDNO. 346) kdevidlivqkekriekvkakkkklekkveegvsglkkh (SEQ ID NO. 347)kvkakkkklckkveegvsglkkh (SEQ ID NO. 348) kakkkklekkvecgvsglkkh (SEQ IDNO. 349) kkkklekkveegvsglkkh (SEQ ID NO. 350) kkklekkveegvsglkkh (SEQ IDNO. 351) kklekkveegvsglkkh (SEQ ID NO. 352) klekkveegvsglkkh (SEQ ID NO.353) kkveegvsglkkh (SEQ ID NO. 354) kveegvsglkkh (SEQ ID NO. 355)hveqnvyvdvdvpamkdqflgilneagglkemffnledvfksesdvitveeikdepvqk (SEQ ID NO.356) hikgleeddleevddlkgsildmlkgdmelgdmdkesledvttklgerveslk (SEQ ID NO.357) hikgleeddleevddlkgsildmlkgdmelgdmdkesledvttk (SEQ ID NO. 358)hikgleeddleevddlkgsildmlkgdmelgdmdk (SEQ ID NO. 359)hikgleeddleevddlkgsildmlk (SEQ ID NO. 360)hiisgdadvlssalgmdccqmktrkkaqrpk (SEQ ID NO. 361) hditttldevvelkdveedkick(SEQ ID NO. 362) kkleevhelk (SEQ ID NO. 363) kleevhelk (SEQ ID NO. 364)ktietdileekkkeiekdh (SEQ ID NO. 365) kkeiekdhfek (SEQ ID NO. 366) kdhfek(SEQ ID NO. 367) kfeeeaeeikh (SEQ ID NO. 368) c) 28 KDA ookinete surfaceantigen precursor Replikins:kdgdtkctlecaqgkkcikhksdhnhksdhnhksdpnhkkknnnnnk (SEQ ID NO. 369)kdgdtkctlecaqgkkcikhksdhnhksdhnhksdpnhkk (SEQ ID NO. 370)kdgdtkctlecaqgkkcikhksdhnhksdhnhksdpnhk (SEQ ID NO. 371)kdgdtkctlecaqgkkcikhksdhnhksdhnhk (SEQ ID NO. 372)kdgdtkctlecaqgkkcikhksdhnhk (SEQ ID NO. 373) kdgdtkctlecaqgkkcikhk (SEQID NO. 374) kdgdtkctleeaqgkk (SEQ ID NO. 375) kdgdtkctlecaqgk (SEQ IDNO. 376) kciqaecnykecgeqkcvwdgih (SEQ ID NO. 377) kecgeqkcvwdgih (SEQ IDNO. 378) hieckcnndyvltnryecepknkctsledtnk (SEQ ID NO. 379) d) Bloodstage trophozoites and schizonts Replikins:ksdhnhksdhnhksdhnhksdhnhksdpnhkkknnnnnk (SEQ ID NO. 380)ksdhnhksdhnhksdhnhksdphnkkknnnnnk (SEQ ID NO. 381)ksdhnhksdhnhksdpnhkkknnnnnk (SEQ ID NO. 382) ksdhnhksdpnhkkknnnnnk (SEQID NO. 383) kkknnnnnkdnksdpnhk (SEQ ID NO. 384) kknnnnnkdnksdpnhk (SEQID NO. 385) knnnnnkdnksdpnhk (SEQ ID NO. 386) kdnksdpnhk (SEQ ID NO.387) ksdpnhk (SEQ ID NO. 388) hslyalqqneeyqkvknekdqneikkikqlieknk (SEQID NO. 389) hslyalqqneeyqkvknekdqneikkik (SEQ ID NO. 390)hslyalqqneeyqkvknekdqneikk (SEQ ID NO. 391) hslyalqqneeyqkvknekdqneik(SEQ ID NO. 392) hklenleemdk (SEQ ID NO. 393) khfddntneqk (SEQ ID NO.394) kkeddekh (SEQ ID NO. 395) keennkkeddekh (SEQ ID NO. 396)ktssgilnkeennkkeddekh (SEQ ID NO. 397) knihikk (SEQ ID NO. 398)hikkkegidigyk (SEQ ID NO. 399) kkmwtcklwdnkgneitknih (SEQ ID NO. 400)kkgiqwnllkkmwtcklwdnkgneitknih (SEQ ID NO. 401)kekkdsnenrkkkqkedkknpnklkkieytnkithffkaknnkqqnnvth (SEQ ID NO. 402)kkdsnenrkkkqkedkknpnklkkieytnkithffkaknnkqqnnvth (SEQ ID NO. 403)kdsnenrkkkqkedkknpnklkkieytnkithffkaknnkqqnnvth (SEQ ID NO. 404)kkqkedkknpnklkkieytnkithffkaknnkqqnnvth (SEQ ID NO. 405)kqkedkknpnklkkieytnkithffkaknnkqqnnvth (SEQ ID NO. 406)kedkknpnklkkieytnkithffkaknnkqqnnvth (SEQ ID NO. 407)knpnklkkieytnkithffkaknnkqqnnvth (SEQ ID NO. 408)kkieytnkithffkaknnkqqnnvth (SEQ ID NO. 409) kieytnkithffkaknnkqqnnvth(SEQ ID NO. 410) kithffkaknnkqqnnvth (SEQ ID NO. 411)hknnedikndnskdikndnskdikndnskdikndnnedikndnskdik (SEQ ID NO. 412)hknnedikndnskdikndnskdikndnskdikndnnedikndnsk (SEQ ID NO. 413)hknnedikndnskdikndnskdikndnskdikndnnedik (SEQ ID NO. 414)hknnedikndnskdikndnskdikndnskdik (SEQ ID NO. 415)hknnedikndnskdikndnskdikndnsk (SEQ ID NO. 416) hknnedikndnskdikndnskdik(SEQ ID NO. 417) hknnedikndnskdikndnsk (SEQ ID NO. 418) hknnedikndnskdik(SEQ ID NO. 419) hknnedik (SEQ ID NO. 420)kkyddlqnkynilnklknsleekneelkkyh (SEQ ID NO. 421)kyddlqnkynilnklknsleekneelkkyh (SEQ ID NO. 422) kynilnklknsleekneelkkyh(SEQ ID NO. 423) klknsleekneelkkyh (SEQ ID NO. 424) knsleekneelkkyh (SEQID NO. 425) kneelkkyh (SEQ ID NO. 426)hmgnnqdinenvynikpqefkeeeeedismvntkk (SEQ ID NO. 427) knsnelkrindnffklh(SEQ ID NO. 428) kpclykkckisqclykkckisqvwwcmpvkdtfntyernnvlnskienniekiph(SEQ ID NO. 429) hinneytnknpkncllykneernyndnnikdyinsmnfkk (SEQ ID NO.430) hinneytnknpkncllykneernyndnnikdyinsmnfk (SEQ ID NO. 431)hinneytnknpkncllyk (SEQ ID NO. 432) knktnqskgvkgeyekkketngh (SEQ ID NO.433) ktnqskgvkgeyekkketngh (SEQ ID NO. 434) kgvkgeyekkketngh (SEQ ID NO.435) kgeyekkketngh (SEQ ID NO. 436) ksgmytnegnkscecsykkkssssnkvh (SEQ IDNO. 437) kscecsykkkssssnkvh (SEQ ID NO. 438) kkkssssnkvh (SEQ ID NO.439) kkssssnkvh (SEQ ID NO. 440) kssssnkvh (SEQ ID NO. 441)himlksgmytnegnkscecsykkkssssnk (SEQ ID NO. 442) himlksgmytnegnkscecsykkk(SEQ ID NO. 443) himlksgmytnegnkscecsykk (SEQ ID NO. 444)himlksgmytnegnkscecsyk (SEQ ID NO. 445)kplaklrkrektqinktkyergdviidnteiqkiiirdyhetlnvhkldh (SEQ ID NO. 446)krektqinktkyergdviidnteiqkiiirdyhetlnvhkldh (SEQ ID NO. 447)ktqinktkyergdviidnteiqkiiirdyhetlnvhkldh (SEQ ID NO. 448)kplaklrkrektqinktkyergdviidnteiqkiiirdyhetlnvh (SEQ ID NO. 449)kplaklrkrektqinktkyergdviidnteiqkiiirdyh (SEQ ID NO. 450)klrkrektqinktkyergdviidnteiqkiiirdyh (SEQ ID NO. 451)krektqinktkyergdviidnteiqkiiirdyh (SEQ ID NO. 452)ktqinktkyergdviidnteiqkiiirdyh (SEQ ID NO. 453)kkdkekkkdsnenrkkkqkedkknpndnklkkieytnkith (SEQ ID NO. 454)kdkekkkdsnenrkkkqkedkknpndnklkkieytnkith (SEQ ID NO. 455)kekkkdsnenrkkkqkedkknpndnklkkieytnkith (SEQ ID NO. 456)kkkdsnenrkkkqkedkknpndnklkkieytnkith (SEQ ID NO. 457)kkdsnenrkkkqkedkknpndnklkkieytnkith (SEQ ID NO. 458)kdsnenrkkkqkedkknpndnklkkieytnkith (SEQ ID NO. 459)kkkqkedkknpndnklkkieytnkith (SEQ ID NO. 460) kkqkedkknpndnklkkieytnkith(SEQ ID NO. 461) kqkedkknpndnklkkieytnkith (SEQ ID NO. 462)kcdkknpndnklkkieytnkith (SEQ ID NO. 463) kknpndnklkkieytnkith (SEQ IDNO. 464) knpndnklkkieytnkith (SEQ ID NO. 465) klkkieytnkith (SEQ ID NO.466) kkieytnkith (SEQ ID NO. 467) kieytnkith (SEQ ID NO. 468)hgqikiedvnnenfnneqmknkyndeekmdiskskslksdflek (SEQ ID NO. 469)hgqikiedvnnenfnneqmknkyndeekmdiskskslk (SEQ ID NO. 470)hgqikiedvnnenfnneqmknkyndeekmdisksk (SEQ ID NO. 471)hgqikiedvnnenfnneqmknkyndeekmdisk (SEQ ID NO. 472)kkyddlqnkynilnklknsleekneelkkyh (SEQ ID NO. 473)kyddlqnkynilnklknsleekneelkkyh (SEQ ID NO. 474) kynilnklknsleekneelkkyh(SEQ ID NO. 475) klknsleekneelkkyh (SEQ ID NO. 476) knsleekneelkkyh (SEQID NO. 477) kneelkkyh (SEQ ID NO. 478)hmgnnqdinenvynikpqefkeeeeedismvntkkcddiqenik (SEQ ID NO. 479)ktnlyniynnknddkdnildnenreglylcdvmknsnelkrindnffklh (SEQ ID NO. 480)knsnelkrindnffklh (SEQ ID NO. 481) krindnffklh (SEQ ID NO. 482)hinneytnknpkncllykneernyndnnikdyinsmnfkk (SEQ ID NO. 483)hinneytnknpkncllykneernyndnnikdyinsmnfk (SEQ ID NO. 484)hinneytnknpkncllyk (SEQ ID NO. 485)kpclykkckisqvwwcmpvkdtfntyernnvlnskienniekiph (SEQ ID NO. 486)kckisqvwwcmpvkdtfntyernnvlnskienniekiph (SEQ ID NO. 487) kienniekiph(SEQ ID NO. 488) knktngskgvkgeyekkketngh (SEQ ID NO. 489)ktngskgvkgeyekkketngh (SEQ ID NO. 490) kgvkgeyekkketngh (SEQ ID NO. 491)kgeyekkketngh (SEQ ID NO. 492)ktiekinkskswffeeldeidkplaklrkrektqinktkyergdviidntciqkiirdyh (SEQ ID NO.493) kinkskswffeeldeidkplaklrkrektqinktkyergdviidnteiqkiirdyh (SEQ IDNO. 494) kplaklrkrektqinktkyergdviidnteiqkiirdyh (SEQ ID NO. 495)himlksqmytnegnkscecsykkkssssnkvh (SEQ ID NO. 496)klrkrektqinktkyergdviidnteiqkiirdyh (SEQ ID NO. 497)krektqinktkyergdviidnteiqkiirdyh (SEQ ID NO. 498)ktqinktkyergdviidnteiqkiirdyh (SEQ ID NO. 499)kplaklrkrektqinktkyergdviidnteiqkiirdyhtlnvhkldh (SEQ ID NO. 500)klrkrektqinktkyergdviidnteiqkiirdyhtlnvhkldh (SEQ ID NO. 501)krektqinktkyergdviidnteiqkiirdyhtlnvhkldh (SEQ ID NO. 502)ktqinktkyergdviidnteiqkiirdyhtlnvhkldh (SEQ ID NO. 503)kplaklrkrektqinktkyergdviidnteiqkiirdyhtlnvh (SEQ ID NO. 504)klrkrektqinktkyergdviidnteiqkiirdyhtlnvh (SEQ ID NO. 505)krektqinktkyergdviidnteiqkiirdyhtlnvh (SEQ ID NO. 506)ktqinktkyergdviidnteiqkiirdyhtlnvh (SEQ ID NO. 507)himlksqmytnegnkscecsykkkssssnkvh (SEQ ID NO. 508)ksqmytnegnkscecsykkkssssnkvh (SEQ ID NO. 509) kscecsykkkssssnkvh (SEQ IDNO. 510) kkkssssnkvh (SEQ ID NO. 511) kkssssnkvh (SEQ ID NO. 512)kssssnkvh (SEQ ID NO. 513) himlksqmytnegnkscecsykkkssssnk (SEQ ID NO.514) himlksqmytnegnkscecsykkk (SEQ ID NO. 515) himlksqmytnegnkscecsykk(SEQ ID NO. 516) himlksqmytnegnkscecsyk (SEQ ID NO. 517)hnnhniqiykdkrinfmnphkvmyhdnmsknertek (SEQ ID NO. 518)hnnhniqiykdkrinfmnphkvmyhdnmsk (SEQ ID NO. 519) hnnhnniqiykdkrinfmnphk(SEQ ID NO. 520) hkvmyhdnmsknertek (SEQ ID NO. 521) hkvmyhdnmsk (SEQ IDNO. 522)

Replikins in Structural Proteins

It has also been determined that some structural proteins includeReplikin structures. Structural proteins are molecules involved intissue and organ support, such as collagen in skin and connective tissueand in membrane structures, for example amyloid A4 precursor protein(APP) in brain. Overproduction of these proteins is associated withdisease; specifically, scleroderma in the case of overproduction ofcollagen in skin (Table 9) and Alzheimer's Disease in the case ofoverproduction of APP in the brain (Table 10).

The association of scleroderma and malignancy has been a source ofcontroversy during recent years. Several mechanisms of interrelationshiphave been suggested in earlier reports. Recent long-term studies suggestan increased association-ratio of scleroderma and malignancy. However,the underlying mechanisms remain elusive. (Wenzel, J. Eur. J. Dermatol.20002 May-June; 12(3): 296-300).

Several proteins concerned with the excessive production of proteins inscleroderma have been found to contain Replikin structures. Thus, theseprovide further examples of unrecognized targets for inhibition orcessation of excessive collagen production. Table 9 provides a list ofproteins in scleroderma and the associated Replikins.

The APP protein is the source of the amyloid beta A4 protein, which inexcessive amounts forms placques in the extracellular spaces in thebrain, producing toxic effects associated with nerve cell loss inAlzheimer's Disease. Most studies to date have focused on the inabilityto clear the excessive deposits of A4, but have not considered that,rather than a waste clearance problem, this may actually be a problem ofoverproduction of the precursor protein APP. The high concentration ofthe Replikins in APP (3.3 Replikins per 100 amino acids) stronglysuggest that overproduction may well be the cause of Alzheimer's Disease(Table 10). Therefore, the Replikins contained in Table 10 can beblocked or inhibited by the same methods as illustrated in detail forthe glioma Replikin.

TABLE 9 Proteins overproduced in scleroderma and associated Replikins:PMC1 HUMAN: hreictiqssggimllkdqvlrcskiagvkvaeitelilk (SEQ ID NO. 523)hreictiqssggimllkdqvlresk (SEQ ID NO. 524) 34KD nucleolar sclerodermaantigen: hreictiqssggimllkdqvlrcskiagvkvaeiteliklkalendqk (SEQ ID NO.525) hreictiqssggirmlkdqvlrcskiagvkvaeitelilk (SEQ ID NO. 526)Fibrillarin: kkmqqenmkqpeqltlepyerdh (SEQ ID NO. 527)kmqqenmkpqeqltlepyerdh (SEQ ID NO. 528) SPOP HUMAN:hemeeskknrveindvepevfkemmcfiytgkapnldk (SEQ ID NO. 529)hemeeskknrveindvepevfkemmcfiytgk (SEQ ID NO. 530) Centromere protein C:khgelkvyk (SEQ ID NO. 531) klilgpqeekgkqh (SEQ ID NO. 532) hnrihhk (SEQID NO. 533) hhnssrkstkktnqssk (SEQ ID NO. 534) hnssrkstkktnqssk (SEQ IDNO. 535) khhnilpktlandkhshkph (SEQ ID NO. 536) hhnilpktlandkhshk (SEQ IDNO. 537) hnilpktlandkhshk (SEQ ID NO. 538) hnilpktlandk (SEQ ID NO. 539)kntpdskkissrnindhh (SEQ ID NO. 540) kntpdskkissrnindh (SEQ ID NO. 541)kdtciqspskecqkshpksvpvsskkk (SEQ ID NO. 542) kdtciqspskecqkshpksvpvsskk(SEQ ID NO. 543) hpksvpvsskkk (SEQ ID NO. 544) hpksvpvsskk (SEQ ID NO.545) hpksvpvssk (SEQ ID NO. 546) Factor CTCBF, KU antigen:kalqekveikqlnh (SEQ ID NO. 547)ktlfplieakkdqvtageifgdnhedgptakklktegggah (SEQ ID NO. 548)ktlfplieakkkdqvtageifqdnb (SEQ ID NO. 549) klcvfkkierhsih (SEQ ID NO.550) klcvfkkierh (SEQ ID NO. 551) kgpsfplkgiteqqkegleivk (SEQ ID NO.552) hgpsfplkgiteqqk (SEQ ID NO. 553) ATP synthase subunit 6:htllkilstflfk (SEQ ID NO. 554) hllgnndknllpsk (SEQ ID NO. 555) FBRLnuclear protein: hrhegvficrgkedalvtk (SEQ ID NO. 556) hegvficrgkedalvtk(SEQ ID NO. 557) hsggnrgrgrggkrghqsgk (SEQ ID NO. 558) krgnqsgknvmveph(SEQ ID NO. 559) krgnqsgknvmvephrh (SEQ ID NO. 560)kkmqqenmkpqeqltlepyerdh (SEQ ID NO. 561) kmqqenmkpqeqltlepyerdh (SEQ IDNO. 562) HP1Hs-alpha protein: haypcdaemkeketak (SEQ ID NO. 563)keanvkcpqiviafyeerltwh (SEQ ID NO. 564) kvldrrvvkgqveyllkwkgfseeh (SEQID NO. 565) kgqveyllkwkgfseeh (SEQ ID NO. 566) FM/Scl nucleolar protein:ksevaagvkksglpsaerlenvlfgphdcsh (SEQ ID NO. 567)ksevaagvkksgplpsaerlenvlfgph (SEQ ID NO. 568)kaaeygkkaksetfrllhakniirpqlk (SEQ ID NO. 569) kaaeygkkaksetfrllhak (SEQID NO. 570) ksetfrllhak (SEQ ID NO. 571) hakniirpqlk (SEQ ID NO. 572)hmnlkiaeelpk (SEQ ID NO. 573) hsldhllklycnvdsnk (SEQ ID NO. 574)hllklycnvdsnk (SEQ ID NO. 575)

TABLE 10 Amyloid beta A4 precursor protein (APP) Replikins: kakerleakh(SEQ ID NO. 576) kdrqhtlk (SEQ ID NO. 577) kdrqhtlkh (SEQ ID NO. 578)ketcsekstnlh (SEQ ID NO. 579) kteeisevkmdaefgh (SEQ ID NO. 580)kteeisevkmdaefghdsgfevrh (SEQ ID NO. 581) kkyvraeqkdrqhtlkh (SEQ ID NO.582) kyvraeqkdrqhtlkh (SEQ ID NO. 583) kkyvraeqkdrqh (SEQ ID NO. 584)kyvraeqkdrqht (SEQ ID NO. 585) hhvfnmlkkyvraeqk (SEQ ID NO. 586)hvfnmlkkyvraeqk (SEQ ID NO. 587) hhvfnmlkkyvraeqkdrqhtlkh (SEQ ID NO.588) hvfnmlkkyvraeqkdrqhtlkh (SEQ ID NO. 589) hahfqkakerleakh (SEQ IDNO. 590) hahfqkakerleak (SEQ ID NO. 591) hfqkakerleak (SEQ ID NO. 592)hqermdvcethlhwhtvaketcsekstnlh (SEQ ID NO. 593)hqermdvcethlhwhtvaketcsek (SEQ ID NO. 594) hwhtvaketcsek (SEQ ID NO.595) htvaketcsek (SEQ ID NO. 596) hlhwhtvaketcsek (SEQ ID NO. 597)hmnvqngkwesdpsgtktcigtk (SEQ ID NO. 598) hmnvqngkwesdpsgtk (SEQ ID NO.599)

Passive Immunity

In another embodiment of the invention, isolated Replikin peptides maybe used to generate antibodies, which may be used, for example toprovide passive immunity in an individual. Passive immunity to thestrain of influenza identified by the method of the invention to be themost likely cause of future influenza infections may be obtained byadministering antibodies to Replikin sequences of the identified strainof influenza virus to patients in need. Similarly, passive immunity tomalaria may be obtained by administering antibodies to Plasmodiumfalciparum Replikin(s).

Various procedures known in the art may be used for the production ofantibodies to Replikin sequences. Such antibodies include but are notlimited to polyclonal, monoclonal, chimeric, humanized, single chain,Fab fragments and fragments produced by an Fab expression library.Antibodies that are linked to a cytotoxic agent may also be generated.Antibodies may also be administered in combination with an antiviralagent. Furthermore, combinations of antibodies to different Replikinsmay be administered as an antibody cocktail.

For the production of antibodies, various host animals or plants may beimmunized by injection with a Replikin peptide or a combination ofReplikin peptides, including but not limited to rabbits, mice, rats, andlarger mammals.

Monoclonal antibodies to Replikins may be prepared by using anytechnique that provides for the production of antibody molecules. Theseinclude but are not limited to the hybridoma technique originallydescribed by Kohler and Milstein, (Nature, 1975, 256:495-497), the humanB-cell hybridoma technique (Kosbor et al., 1983, Immunology Today,4:72), and the EBV hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Inaddition, techniques developed for the production of chimeric antibodies(Morrison et al., 1984, Proc. Nat. Acad. Sci USA, 81:6851-6855) or othertechniques may be used. Alternatively, techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778) can beadapted to produce Replikin-specific single chain antibodies.

Particularly useful antibodies of the invention are those thatspecifically bind to Replikin sequences contained in peptides and/orpolypeptides of influenza virus. For example, antibodies to any ofpeptides observed to be present in an emerging or re-emerging strain ofinfluenza virus and combinations of such antibodies are useful in thetreatment and/or prevention of influenza. Similarly, antibodies to anyReplikins present on malaria antigens and combinations of suchantibodies are useful in the prevention and treatment of malaria.

Antibody fragments which contain binding sites for a Replikin may begenerated by known techniques. For example, such fragments include butare not limited to F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecules and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries can be generated (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

The fact that antimalignin antibody is increased in concentration inhuman malignancy regardless of cancer cell type (FIG. 5), and that thisantibody binds to malignant cells regardless of cell type now may beexplained by the presence of the Replikin structures herein found to bepresent in most malignancies (FIG. 1 and Table 2). Population studieshave shown that antimalignin antibody increases in concentration inhealthy adults with age, and more so in high-risk families, as thefrequency of cancer increases. An additional two-fold or greaterantibody increase which occurs in early malignancy has beenindependently confirmed with a sensitivity of 97% in breast cancers 1-10mm in size. Shown to localize preferentially in malignant cells in vivo,histochemically the antibody does not bind to normal cells butselectively binds to (FIG. 4 a, b) and is highly cytotoxic totransformed cells in vitro (FIG. 4 c-f). Since in these examples thesame antibody is bound by several cell types, that is, brain glioma,hematopoietic cells (leukemia), and small cell carcinoma of lung,malignant Replikin class unity is again demonstrated.

Antimalignin does not increase with benign proliferation, butspecifically increases only with malignant transformation andreplication in breast in vivo and returns from elevated to normal valuesupon elimination of malignant cells (FIG. 5). Antimalignin antibodyconcentration has been shown to relate quantitatively to the survival ofcancer patients, that is, the more antibody, the longer the survival.Taken together, these results suggest that anti-Replikin antibodies maybe a part of a mechanism of control of cell transformation andreplication. Augmentation of this immune response may be useful in thecontrol of replication, either actively with synthetic Replikins asvaccines, or passively by the administration of anti-Replikinantibodies, or by the introduction of non-immune based organic agents,such as for example, carbohydrates, lipids and the like, which aresimilarly designed to target the Replikin specifically.

In another embodiment of the invention, immune serum containingantibodies to one or more Replikins obtained from an individual exposedto one or more Replikins may be used to induce passive immunity inanother individual or animal. Immune serum may be administered via i.v.to a subject in need of treatment. Passive immunity also can be achievedby injecting a recipient with preformed antibodies to one or moreReplikins. Passive immunization may be used to provide immediateprotection to individuals who have been exposed to an infectiousorganism. Administration of immune serum or preformed antibodies isroutine and the skilled practitioner can readily ascertain the amount ofserum or antibodies needed to achieve the desired effect.

Synthetic Replikin Vaccine Active Immunity

Synthetic Replikin vaccines, based on Replikins such as the gliomaReplikin (SEQ ID NO.: 1) “kagvaflhkk” or the hepatitis C Replikin (SEQID NO.: 18) “hyppkpgcivpak”, or HIV Replikins such as (SEQ ID NO.: 5)“kcfncgkegh” or (SEQ ID NO.: 6) “kvylawvpahk” or preferably, aninfluenza vaccine based on conserved and/or emerging or re-emergingReplikin(s) over a given time period may be used to augment antibodyconcentration in order to lyse the respective virus infected cells andrelease virus extracellularly where chemical treatment can then beeffective. Similarly, a malaria vaccine, based on Replikins observed inPlasmodium falciparum malaria antigens on the merozoite surface orwithin the parasitophorous vacuole, for example, can be used to generatecytotoxic antibodies to malaria.

Recognin and/or Replikin peptides may be administered to a subject toinduce the immune system of the subject to produce anti-Replikinantibodies. Generally, a 0.5 to about 2 mg dosage, preferably a 1 mgdosage of each peptide is administered to the subject to induce animmune response. Subsequent dosages may be administered if desired.

The Replikin sequence structure is associated with the function ofreplication. Thus, whether the Replikins of this invention are used fortargeting sequences that contain Replikins for the purpose of diagnosticidentification, promoting replication, or inhibiting or attackingreplication, for example, the structure-function relationship of theReplikin is fundamental.

It is preferable to utilize only the specific Replikin structure whenseeking to induce antibodies that will recognize and attach to theReplikin fragment and thereby cause destruction of the cell. Even thoughthe larger protein sequence may be known in the art as having a“replication associated function,” vaccines using the larger proteinoften have failed or proven ineffective.

Although the present inventors do not wish to be held to a singletheory, the studies herein suggest that the prior art vaccines areineffective because they are based on the use of the larger proteinsequence. The larger protein sequence invariably has one or moreepitopes (independent antigenic sequences that can induce specificantibody formation); Replikin structures usually comprise one of thesepotential epitopes. The presence of other epitopes within the largerprotein may interfere with adequate formation of antibodies to theReplikin, by “flooding” the immune system with irrelevant antigenicstimuli that may preempt the Replikin antigens, See, e.g., Webster, R.G., J. Immunol., 97(2):177-183 (1966); and Webster et al., J. Infect.Dis., 134:48-58, 1976; Klenerman et al, Nature 394:421-422 (1998) for adiscussion of this well-known phenomenon of antigenic primacy wherebythe first peptide epitope presented and recognized by the immune systemsubsequently prevails and antibodies are made to it even though otherpeptide epitopes are presented at the same time. This is another reasonthat, in a vaccine formulation, it is important to present the constantReplikin peptide to the immune system first, before presenting otherepitopes from the organism so that the Replikin is not preempted butlodged in immunological memory.

The formation of an antibody to a non-Replikin epitope may allow bindingto the cell, but not necessarily lead to cell destruction. The presenceof structural “decoys” on the C-termini of malaria proteins is anotheraspect of this ability of other epitopes to interfere with binding ofeffective anti-Replikin antibodies, since the decoy epitopes have manylysine residues, but no histidine residues. Thus, decoy epitopes maybind anti-Replikin antibodies, but may keep the antibodies away fromhistidine-bound respiratory enzymes. Treatment may therefore be mostefficacious in two stages: 1) proteases to hydrolize decoys, then; 2)anti-Replikin antibodies or other anti-Replikin agents.

It is well known in the art that in the course of antibody productionagainst a “foreign” protein, the protein is first hydrolyzed intosmaller fragments. Usually fragments containing from about six to tenamino acids are selected for antibody formation. Thus, if hydrolysis ofa protein does not result in Replikin-containing fragments,anti-Replikin antibodies will not be produced. In this regard, it isinteresting that Replikins contain lysine residues located six to tenamino acids apart, since lysine residues are known to bind to membranes.

Furthermore, Replikin sequences contain at least one histidine residue.Histidine is frequently involved in binding to redox centers. Thus, anantibody that specifically recognizes a Replikin sequence has a betterchance of inactivating or destroying the cell in which the Replikin islocated, as seen with anti-malignin antibody, which is perhaps the mostcytotoxic anti-cancer antibody yet described, being active at picogramsper cell.

One of the reasons that vaccines directed towards a particular proteinantigen of a disease causing agent have not been fully effective inproviding protection against the disease (such as foot and mouth vaccinewhich has been developed against the VP1 protein or large segments ofthe VP1 protein) is that the best antibodies have not been produced,that is—it is likely that the antibodies to the Replikins have not beenproduced. Replikins have not been produced. That is, either epitopesother than Replikins present in the larger protein fragments mayinterfere according to the phenomenon of antigenic primacy referred toabove, and/or because the hydrolysis of larger protein sequences intosmaller sequences for processing to produce antibodies results in lossof integrity of any Replikin structure that is present, e.g., theReplikin is cut in two and/or the histidine residue is lost in thehydrolytic processing. The present studies suggest that for an effectivevaccine to be produced, the Replikin sequences, and no other epitope,should be used as the vaccine. For example, a vaccine of the inventioncan be generated using any one of the Replikin peptides identified bythe three point recognition system.

Particularly preferred peptides—for example—an influenza vaccine includepeptides that have been demonstrated to be conserved over a period ofone or more years, preferably about three years or more, and/or whichare present in a strain of influenza virus shown to have the highestincrease in concentration of Replikins relative to Replikinconcentration in other influenza virus strains, e.g., an emergingstrain. The increase in Replikin concentration preferably occurs over aperiod of at least about six months to one year, preferably at leastabout two years or more, and most preferably about three years or more.Among the preferred Replikin peptides for use in an influenza virusvaccine are those Replikins observed to “re-emerge” after an absencefrom the hemagglutinin amino acid sequence for one or more years.

The Replikin peptides of the invention, alone or in various combinationsare administered to a subject, preferably by i.v. or intramuscularinjection, in order to stimulate the immune system of the subject toproduce antibodies to the peptide. Generally the dosage of peptides isin the range of from about 0.1 μg to about 10 mg, preferably about 10 μgto about 1 mg, and most preferably about 50 μg to about 500 ug. Theskilled practitioner can readily determine the dosage and number ofdosages needed to produce an effective immune response.

Quantitative Measurement Early Response(s) to Replikin Vaccines

The ability to measure quantitatively the early specific antibodyresponse in days or a few weeks to a Replikin vaccine is a majorpractical advantage over other vaccines for which only a clinicalresponse months or years later can be measured.

Adjuvants

Various adjuvants may be used to enhance the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels, such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, key limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG andCorynebacterium parvum.

Replikin Nucleotide Sequences

Replikin DNA or RNA may have a number of uses for the diagnosis ofdiseases resulting from infection with a virus, bacterium or otherReplikin encoding agent. For example, Replikin nucleotide sequences maybe used in hybridization assays of biopsied tissue or blood, e.g.,Southern or Northern analysis, including in situ hybridization assays,to diagnose the presence of a particular organism in a tissue sample oran environmental sample, for example. The present invention alsocontemplates kits containing antibodies specific for particularReplikins that are present in a particular pathogen of interest, orcontaining nucleic acid molecules (sense or antisense) that hybridizespecifically to a particular Replikin, and optionally, various buffersand/or reagents needed for diagnosis.

Also within the scope of the invention are oligoribonucleotidesequences, that include antisense RNA and DNA molecules and ribozymesthat function to inhibit the translation of Replikin- orrecognin-containing mRNA. Both antisense RNA and DNA molecules andribozymes may be prepared by any method known in the art. The antisensemolecules can be incorporated into a wide variety of vectors fordelivery to a subject. The skilled practitioner can readily determinethe best route of delivery, although generally i.v. or i.m. delivery isroutine. The dosage amount is also readily ascertainable.

Particularly preferred antisense nucleic acid molecules are those thatare complementary to a Replikin sequence contained in a mRNA encoding,for example, an influenza virus polypeptide, wherein the Replikinsequence comprises from 7 to about 50 amino acids including:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.        More preferred are antisense nucleic acid molecules that are        complementary to a Replikin present in the coding strand of the        gene or to the mRNA encoding the influenza virus hemagglutinin        protein, wherein the antisense nucleic acid molecule is        complementary to a nucleotide sequence encoding a Replikin that        has been demonstrated to be conserved over a period of six        months to one or more years and/or which are present in a strain        of influenza virus shown to have an increase in concentration of        Replikins relative to Replikin concentration in other influenza        virus strains. The increase in Replikin concentration preferably        occurs over a period of at least six months, preferably about        one year, most preferably about two or three years or more.

Similarly, antisense nucleic acid molecules that are complementary tomRNA those that are complementary to a mRNA encoding bacterial Replikinscomprising a Replikin sequence of from 7 to about 50 amino acidsincluding:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.        More preferred are antisense nucleic acid molecules that are        complementary to the coding strand of the gene or to the mRNA        encoding a protein of the bacteria.

Diagnostic Applications

For organisms such as diatom plankton, foot and mouth disease virus,tomato leaf curl gemini virus, hepatitis B and C, HIV, influenza virusand malignant cells, identified constituent Replikins are useful asvaccines, and also may be usefully targeted for diagnostic purposes. Forexample, blood collected for transfusions may be screened forcontamination of organisms, such as HIV, by screening for the presenceof Replikins shown to be specific for the contamination organism. Also,screening for Replikin structures specific for a particular pathologicalorganism leads to diagnostic detection of the organism in body tissue orin the environment.

Replikin Stimulation of Growth

In another embodiment of the invention, Replikin structures are used toincrease the replication rate of cells, tissues or organs. A method isavailable to increase replication rates by the addition of specificReplikin structures for other cells, tissues or organs that it isdesired to replicate more rapidly, together with or without appropriatestimulae to cell division know in the art for said cells, tissues ororgans to increase the rate of replication and yield. This may beaccomplished, for example, by methods known in the art, by modifying ortransforming a gene encoding for or associated with a protein or enzymehaving a replication function in the organism with at least one Replikinstructure.

In another aspect of the invention, Replikin structures are used toincrease the replication of organisms. The present inventiondemonstrates that in influenza virus, for example, increased replicationassociated with epidemics is associated with increased concentration ofReplikins. The increase is due to 1) the reappearance of particularReplikin structures, which were present in previous years, but whichthen disappeared for one or more years; and/or 2) by the appearance ofnew Replikin compositions. In addition, in malaria Replikins, repetitionof the same Replikin in a single protein occurs.

Thus, the present invention provides methods and compositions forincreasing the replication of organisms. Similarly, in the manner thatReplikins of different organisms can be targeted to inhibit replicationof any organism, Replikins can be used to increase the replication ofany organism. For example, production of rice, maize, and wheat crops,which are critical to feeding large populations in the world, can beimproved, for example, by increasing the concentration (number ofReplikins/100 amino acid residues) of any particular strain of rice.

As an example, in the Oryza sativa strain of rice, catalase isolatedfrom immature seeds was observed to contain the following differentReplikins within the 491 amino acid sequence of the protein:

(SEQ ID NO. 625) kfpdvihafkpnprsh (SEQ ID NO. 626) kfpdvihafk (SEQ IDNO. 627) karyvkfhwk (SEQ ID NO. 628) hpkvspelraiwvnylsqedeslgvkianlnvk(SEQ ID NO. 629) hrdeevdyypsrhaplrhapptpitprpvvgrrqkatihkqndfk (SEQ IDNO. 630) katihkqndfk (SEQ ID NO. 631) happtpitprpvvgrrqkatihkqndfk (SEQID NO. 632) kfrpsssfdtkttttnagapvwndnealtvgprgpilledyhliekvah (SEQ IDNO. 633) kfrpsssfdtkttttnagapvwndnealtvgprgpilledyn

Thus, by using recombinant gene cloning techniques well known in theart, the concentration of Replikin structures in an organism, such as afood crop plant, can be increased, which will promote increasedreplication of the organism. For example, inserting additional Replikinsequences like the Replikins identified above into the Oryza sativacatalase gene by methods well know in the art will promotethisorganism's replication.

Similarly, in the NBS-LRR protein of Oryza sativa (japonica cultivargroup), the following Replikins were found:

(SEQ ID NO. 634) kvkahfqkh (SEQ ID NO. 635) kvkahfqk (SEQ ID NO. 636)kdyeidkddlih (SEQ ID NO. 637) hmkqcfafcavfpkdyeidk (SEQ ID NO. 638)hmkqcfafcavfpk (SEQ ID NO. 639)hvfwelvwrsffqnvkqigsifqrkvyrygqsdvttskihdlmhdlavh (SEQ ID NO. 640)kqigsifqrkvrygpsdvttskihdlmhdlavh (SEQ ID NO. 641)kqigsifqrkvyrygpsdvttskihdlmh (SEQ ID NO. 642) kqigsifqrkvyrygqsdvttskih

Further, for aspartic proteinase oryzasin 1 precursor protein, thefollowing Replikins were found:

khgvsagik (SEQ ID NO. 643) htvfdygkmrvgfak (SEQ ID NO. 644)hsryksgqsstyqkngk (SEQ ID NO. 645)

Similarly, in the MADS-box protein FDRMADS3 transcription factor ofOryza sativa (indica cultivar-group), the following Replikins werefound:

(SEQ ID NO. 646) kqeamvlkqeinllqkglryiygnraneh (SEQ ID NO. 647)kqeinllqkglryiygnraneh (SEQ ID NO. 648)kskegmlkaaneilqekiveqnglidvgmmvadqqngh (SEQ ID NO. 649)kaaneilqekiveqnglidvgmmvadqqngh

Similarly, in LONI MAIZE (ATP-binding redox associated Hydrolase; Serineprotease; Multigene family; Mitochondrion), the following Replikins werefound:

(SEQ ID NO. 650) kvlaahrygik (SEQ ID NO. 651) klkiamkhliprvleqh (SEQ IDNO. 652) klkiamkh (SEQ ID NO. 653) ktslassiakalnrkfirislggvkdeadirgh(SEQ ID NO. 654) kalnrkfirislggvkdeadirgh (SEQ ID NO. 655)kfirislggvkdeadirgh (SEQ ID NO. 656)kvrlskatelvdrhlqsilvaekitqkvegqlsksqk (SEQ ID NO. 657)hlqsilvaekitqkvegglsksqk (SEQ ID NO. 658) kvrlskatelvdrh (SEQ ID NO.659) kvggsavesskqdtkngkepihwhskgvaaralh (SEQ ID NO. 660)kvggsavesskqdtkngkepihwh (SEQ ID NO. 661) kvggsavesskqdtkngkepih (SEQ IDNO. 662) kqdtkngkepihwhskgvaaralh (SEQ ID NO. 663) kqdtkngkepih

Similarly, for Glyceraldehyde 3-phospate dehydrogenase A, a chloroplastprecursor, the following Replikins are found:

hrdlrraraaalnivptstgaakavslvlpnlk (SEQ ID NO. 664) kvlddqkfgiikgtmttth(SEQ ID NO. 665) hiqagakkvlitapgk (SEQ ID NO. 666)hgrgdaspldviaindtggvkqashllk (SEQ ID NO. 667) kqashllk (SEQ ID NO. 697)

Further, examples of rust resistance-like protein RP1-4 (Zea mays) foundinclude the following Replikins:

kvrrvlskdysslkqlmtlmmdddiskhlqiiesgleeredkvwmkeniik (SEQ ID NO. 668)kvrrvlskdysslkqlmtlmmdddiskh (SEQ ID NO. 669) hlqiiesgleeredkvwmkeniik(SEQ ID NO. 670) hdlreniimkaddlask (SEQ ID NO. 671) hvqnlenvigkdealask(SEQ ID NO. 672) kkqgyelrqlkdlnelggslh (SEQ ID NO. 673)kqgyelrqlkdlnelggslh (SEQ ID NO. 674) klylksrlkelilewssengmdamnilh (SEQID NO. 675) hlqllqlngmverlpnkvcnlsklrylrgykdqipnigk (SEQ ID NO. 676)hlqllqlngmverlpnkvcnlskrylrgyk (SEQ ID NO. 677) hlqllqlngmverlpnkvcnlsk(SEQ ID NO. 678) hnsnklpksvgelk (SEQ ID NO. 679) klpkvgelkh (SEQ ID NO.680) hlsvrvesmqkhkeiiyk (SEQ ID NO. 681) khkeiiyk (SEQ ID NO. 682)klrdilqesqkfllvldlalfkh (SEQ ID NO. 683)hafsgaeikdqllrmklqdtaeeiakrlgqcplaakvlgsrmcrrk (SEQ ID NO. 684)hafsgaeikdqllrmk (SEQ ID NO. 685)klqdtacciakrlgqclaakvlgsrmcrrkdiaewkaadvwfeksh (SEQ ID NO. 686)kvlgsrmcrrkdiacwkaadvwfeksh (SEQ ID NO. 687) kdiaewkaadvwfeksh (SEQ IDNO. 688) kaadvwfeksh (SEQ ID NO. 689)hvptttslptskvfgrnsdrdrivkfllgktttaeasstk (SEQ ID NO. 690)kailteakqlrdllglph (SEQ ID NO. 691) kakaksgkgpllredessstattvmkpfh (SEQID NO. 692) ksphrgkleswlrrlkeafydaedlldeh (SEQ ID NO. 693)ksphrgkleswlrrlk (SEQ ID NO. 694) hrgkleswlrrlk (SEQ ID NO. 695) ksphrgk(SEQ ID NO. 696)

As discussed previously, the Replikin in wheat ubiquitin activatingenzyme E (SEQ ID Nos. 601-603) is conserved. This conservation ofReplikin structure provides reliable targets for stimulation of plantgrowth.

The close relationship of Replikins to redox enzymes is also clearlyindicated in this structure in wheat. Thus, this wheat ubiquitinactivating enzyme E activates ubiquitin by first adenylating with ATPits carboxy-terminal glycine residue and, thereafter, linking thisresidue to the side chain of a cysteine residue in E1 (SEQ ID NO. 603),yielding an ubiquitin-E1 thiolester and free AMP.

A further example of the relationship of wheat Replikins to redoxenzymes was also found in the PSABWheat Protein, Photosystem I P700chlorophyll A apoprotein A2 (PsaB) (PSI-B) isolated from bread Chinesespring wheat Chloroplast Triticum aestivum. This protein functions asfollows: PsaA and PsaB bind 9700, the primary electron donor ofphotosystem I (PSI), as well as the electron acceptors A0, A1, and FX.PSI functions as a plastocyanin/cytochrome c6-ferredoxin oxidoreductase.Cofactor P700 is a chlorophyll A dimer, A0 is chlorophyll A, A1 is aphylloquinone and FX is a 4Fe-4S iron-sulfur center. The subunit ApsaA/S heterodimer binds the P700 chlorophyll special pair andsubsequent electron acceptors. The PSI reaction center of higher plantsand algae is composed of one at least 11 subunits. This is an integralmembrane protein of the Chloroplast thylakoid membrane. The 4Fe-4Siron-sulfur “center” to which ‘h’ bind is critical; hence thesignificance of ‘h’ in Replikin structure. Next to bacterial Replikins,these wheat Replikins and plant Replikins are the most primitiveevolutionary illustrations of the importance of the Replikin structureto replication and the energy source needed for replication. This basicrelationship carries through algae, virus Replikins, bacteria, cancercells, and apparently all organisms with regard to replication.

Further examples of Replikins were found in the PSAB Wheat protein,which is critical fox wheat growth. These include:

hlqpkwkpslswfknaesrlnhh (SEQ ID NO. 604) hlqpkwkpslswfk (SEQ ID NO. 605)kwkpslswfknaesrlnhh (SEQ ID NO. 606) kwkpslswfknaesrlnh (SEQ ID NO. 607)kpslswfknaesrlnhh (SEQ ID NO. 608) kpslswfknaesrlnh (SEQ ID NO. 609)hhaialglhtttlilvkgaldargsklmpdkk (SEQ ID NO. 610)haialglhtttlilvkgaldargsklmpdkk (SEQ ID NO. 611)hhaialglhtttlilvkgaldargsk (SEQ ID NO. 612) haialglhtttlilvkgaldargsk(SEQ ID NO. 613) htttlilvkgaldargsklmpdkk (SEQ ID NO. 614)htttlilvkgaldargsklmpdk (SEQ ID NO. 615) htttlilvkgaldargsk (SEQ ID NO.616)

A further example of the relationship of wheat Replikins to redox isprovide in the PSAA_WHEAT Photosystem I 9700 chlorophyll A apoproteinA1, that include:

(SEQ ID NO. 617) hhhlaiailfliaghmyrtnwgighglkdileahkgpftgqghk (SEQ IDNO. 618) hhlaiailfliaghmyrtnwgighglkdileahkgpftgqghk (SEQ ID NO. 619)hlaiailfliaghmyrtnwgighglkdileahkgpftgqghk (SEQ ID NO. 620)hmyrtnwgighglkdileahkgpftgqghk (SEQ ID NO. 621) hglkdileahkgpftgqghk(SEQ ID NO. 622) hdileahkgpftgqghk (SEQ ID NO. 623) hkgpftgqghk (SEQ IDNO. 624) kgpftgqghk

Computer Software for Identifying Replikins

The present invention also provides methods for identifying Replikinsequences in an amino acid or nucleic acid sequence. Visual scanning ofover four thousand sequences was performed in developing the present3-point-recognition methods. However, data banks comprising nucleotideand/or amino acid sequences can also be scanned by computer for thepresence of sequences meeting the 3 point recognition requirements.

According to another embodiment of the invention, three-pointrecognition methods described herein may be performed by a computer.FIG. 6 is a block diagram of a computer available for use with theforegoing embodiments of the present invention. The computer may includea processor, an input/output device and a memory storing executableprogram instructions representing the 3-point-recognition methods of theforegoing embodiments. The memory may include a static memory, volatilememory and/or a nonvolatile memory. The static memory conventionally maybe a read only memory (“ROM”) provided on a magnetic, or an electricalor optical storage medium. The volatile memory conventionally may be arandom accessmemory (“RAM”) and may be integrated as a cache within theprocessor or provided externally from the processor as a separateintegrated circuit. The non-volatile memory may be an electrical,magnetic or optical storage medium.

From a proteomic point of view the construction of a “3-pointrecognition” template based on the new glioma peptide sequence leddirectly to identification of a biology-wide class of proteins havingrelated structures and functions. The operation of the3-point-recognition method resembles identification by the use of a“keyword” search; but instead of using the exact spelling of the keyword“kagvaflhkk” (SEQ ID NO.: 1) as in a typical sequence homology search,or in the nucleotide specification of an amino acid, an abstraction ofthe keyword delimited by the “3-point-recognition” parameters is used.This delimited abstraction, although derived from a single relativelyshort amino acid sequence leads to identification of a class of proteinswith structures that are defined by the same specifications. Thatparticular functions, in this case transformation and replication, inaddition to structures, turn out also to be shared by members of theexposed class suggests that these structures and functions are related.Thus, from this newly identified short peptide sequence, a molecularrecognition ‘language’ has been formulated, which previously has notbeen described. Further, the sharing of immunological specificity bydiverse members of the class, as here demonstrated for the cancerReplikins, suggests that B cells and their product antibodies recognizeReplikins by means of a similar recognition language.

Other Uses of the Three Point Recognition Method

Since “3-point-recognition” is a proteomic method that specifies aparticular class of proteins, using three or more different recognitionpoints for other peptides similarly should provide useful informationconcerning other proteins classes. Further, the “3-point-recognition”method is applicable to other recognins, for example to the TOLL‘innate’ recognition of lipopolyssacharides of organisms. The threepoint recognition method may also be modified to identify other usefulcompounds of covalently linked organic molecules, including othercovalently linked amino acids, nucleotides, carbohydrates, lipids orcombinations thereof. In this embodiment of the invention a sequence isscreened for subsequences containing three or more desired structuralcharacteristics. In the case of screening compounds composed ofcovalently linked amino acids, lipids or carbohydrates the subsequenceof 7 to about 50 covalently linked units should contain (1) at least onefirst amino acid, carbohydrate or lipid residue located seven to tenresidues from a second of the first amino acid, carbohydrate or lipidresidue; (2) encoding at least one second amino acid, lipid orcarbohydrate residue; and (3) at least 6% of the first amino acid,carbohydrate or lipid residue. In the case of screening nucleotidesequences, the subsequence of about 21 to about 150 nucleotides shouldcontain (1) at least one codon encoding a first amino acid locatedwithin eighteen to thirty nucleotides from a second codon encoding thefirst amino acid residue; (2) at least one second amino acid residue;and (3) encodes at least 6% of said first amino acid residue.

Several embodiments of the present invention are specificallyillustrated and described herein. However, it will be appreciated thatmodifications and variations of the present invention are encompassed bythe above teachings and within the purview of the appended claimswithout departing from the spirit and intended scope of the invention.

EXAMPLE 1 Process for Extraction, Isolation and Identification ofReplikins and the Use of Replikins to Target, Label or DestroyReplikin-Containing Organisms

a) Algae

The following algae were collected from Bermuda water sites and eitherextracted on the same day or frozen at −20 degrees C. and extracted thenext day. The algae were homogenized in a cold room (at 0 to 5 degreesC.) in 1 gram aliquots in neutral buffer, for example 100 cc. of 0.005Mphosphate buffer solution, pH 7 (“phosphate buffer”) for 15 minutes in aWaring blender, centrifuged at 3000 rpm, and the supernatantconcentrated by perevaporation and dialyzed against phosphate buffer inthe cold to produce a volume of approximately 15 ml. The volume of thisextract solution was noted and an aliquot taken for protein analysis,and the remainder was fractionated to obtain the protein fraction havinga pK range between 1 and 4.

The preferred method of fractionation is chromatography as follows: Theextract solution is fractionated in the cold room (4 degrees C.) on aDEAE cellulose (Cellex-D) column 2.5×11.0 cm, which has beenequilibrated with 0.005M phosphate buffer. Stepwise eluting solventchanges are made with the following solutions:

-   -   Solution 1-4.04 g. NaH2P04 and 0.5 g NaH2P04 are dissolved in 15        litres of distilled water (0.005 molar, pH 7);    -   Solution 2-8.57 g. NaH2P04 is dissolved in 2,480 ml. of        distilled water;    -   Solution 3-17.1 g. of NaH2P04 is dissolved in 2480 ml of        distilled water (0.05 molar, pH 4.7);    -   Solution 4-59.65 g. of NaH2P04 is dissolved in 2470 ml distilled        water (0.175 molar);    -   Solution 5-101.6 g. of NaH2P04 is dissolved in 2455 ml distilled        water (pH 4.3);    -   Solution 6-340.2 g. of NaH2P04 is dissolved in 2465 of distilled        water (1.0 molar, pX-i 4.1);    -   Solution 7-283.63 g. of 80% phosphoric acid (H3P04) is made up        in 2460 ml of distilled water (1.0 molar, pH 1.0).

The extract solution, in 6 to 10 ml volume, is passed onto the columnand overlayed with Solution 1, and a reservoir of 300 ml of Solution 1is attached and allowed to drip by gravity onto the column. Three mlaliquots of eluant are collected and analyzed for protein content at OD280 until all of the protein to be removed with Solution 1 has beenremoved from the column. Solution 2 is then applied to the column,followed in succession by Solutions 3, 4, 5, 6 and 7 until all of theprotein which can, be removed with each Solution is removed from thecolumn. The eluates from Solution 7 are combined, dialyzed againstphosphate buffer, the protein content determined of both dialysand anddialyzate, and both analyzed by gel electrophoresis. One or two bands ofpeptide or protein of molecular weight between 3,000 and 25,000 Daltonsare obtained in Solution 7. For example the algae Caulerpa mexicana,Laurencia obtura, Cladophexa prolifera, Sargassum natans, Caulerpaverticillata, Halimeda tuna, and Penicillos capitatus, after extractionand treatment as above, all demonstrated in Solution 7 eluates sharppeptide bands in this molecular weight region with no contaminants.These Solution 7 proteins or their eluted bands are hydrolyzed, and theamino acid composition determined. The peptides so obtained, which havea lysine composition of 6% or greater are Replikin precursors. TheseReplikin peptide precursors are then determined for amino acid sequenceand the Replikins are determined by hydrolysis and mass spectrometry asdetailed in U.S. Pat. No. 6,242,578 B1. Those which fulfill the criteriadefined by the “3-point-recognition” method are identified as Replikins.This procedure can also be applied to obtain yeast, bacterial and anyplant Replikins.

b) Virus

Using the same extraction and column chromatography separation methodsas above in a) for algae, Replikens in virus-infected cells are isolatedand identified.

c) Tumor Cells In Vivo and In Vitro Tissue Culture

Using the same extraction and column chromatography separation methodsas above in a) for algae, Replikins in tumor cells are isolated andidentified. For example, Replikin precursors of Astrocytin isolated frommalignant brain tumors, Malignin (Aglyco 1OB) isolated from glioblastomatumor cells in tissue culture, MCF7 mammary carcinoma cells in tissueculture, and P3J Lymphoma cells in tissue culture each treated as abovein a) yielded Replikin precursors with lysine content of 9.1%, 6.7%,6.7%, and 6.5% respectively. Hydrolysis and mass spectrometry of Aglyco1OB as described in Example 10 U.S. Pat. No. 6,242,578 B1 produced theamino acid sequence, ykagvaflhkkndiide the 16-mer Replikin.

EXAMPLE 2

As an example of diagnostic use of Replikins: Aglyco 1OB or the 16-merRepliken may be used as antigen to capture and quantify the amount ofits corresponding antibody present in serum for diagnostic purposes areas shown in FIGS. 2,3,4 and 7 of U.S. Pat. No. 6,242,578 B1.

As an example of the production of agents to attach to Replikins forlabeling, nutritional or destructive purposes: Injection of the 16-merReplikin into rabbits to produce the specific antibody to the 16-merReplikin is shown in Example 6 and FIGS. 9A and 9B of U.S. Pat. No.6,242,578 B1.

As an example of the use of agents to label Replikins: The use ofantibodies to the 16-mer Replikin to label specific cells which containthis Replikin is shown in FIG. 5 and Example 6 of U.S. Pat. No.6,242,578 B1.

As an example of the use of agents to destroy Replikins: The use ofantibodies to the 16-mer Replikin to inhibit or destroy specific cellswhich contain this Replikin is shown in FIG. 6 of U.S. Pat. No.6,242,578 B1.

EXAMPLE 3

Analysis of sequence data of isolates of influenza virus hemagglutininprotein or neuraminidase protein for the presence and concentration ofReplikins is carried out by visual scanning of sequences or through useof a computer program based on the 3-point recognition system describedherein. Isolates of influenza virus are obtained and the amino acidsequence of the influenza hemagglutinin and/or neuraminidase protein isobtained by any art known method, such as by sequencing thehemagglutinin or neuraminidase gene and deriving the protein sequencetherefrom. Sequences are scanned for the presence of new Replikins,conservation of Replikins over time and concentration of Replikins ineach isolate. Comparison of the Replikin sequences and concentrations tothe amino acid sequences obtained from isolates at an earlier time, suchas about six months to about three years earlier, provides data that areused to predict the emergence of strains that are most likely to be thecause of influenza in upcoming flu seasons, and that form the basis forseasonal influenza peptide vaccines or nucleic acid based vaccines.Observation of an increase in concentration, particularly a stepwiseincrease in concentration of Replikins in a given strain of influenzavirus for a period of about six months to about three years or more is apredictor of emergence of the strain as a likely cause of influenzaepidemic or pandemic in the future.

Peptide vaccines or nucleic acid-based vaccines based on the Replikinsobserved in the emerging strain are generated. An emerging strain isidentified as the strain of influenza virus having the highest increasein concentration of Replikin sequences within the hemagglutinin and/orneuraminidase sequence during the time period. Preferably, the peptideor nucleic acid vaccine is based on or includes any Replikin sequencesthat are observed to be conserved in the emerging strain. ConservedReplikins are preferably those Replikin sequences which are present inthe hemagglutinin or neuraminidase protein sequence for about two yearsand preferably longer. The vaccines may include any combination ofReplikin sequences identified in the emerging strain.

For vaccine production, the Replikin peptide or peptides identified asuseful for an effective vaccine are synthesized by any method, includingchemical synthesis and molecular biology techniques, including cloning,expression in a host cell and purification therefrom. The peptides arepreferably admixed with a pharmaceutically acceptable carrier in anamount determined to induce a therapeutic antibody reaction thereto.Generally, the dosage is about 0.1 μg to about 10 mg.

The influenza vaccine is preferably administered to a patient in needthereof prior to the onset of “flu season.” Influenza flu seasongenerally occurs in late October and lasts through late April. However,the vaccine may be administered at any time during the year. Preferably,the influenza vaccine is administered once yearly, and is based onReplikin sequences observed to be present, and preferably conserved inthe emerging strain of influenza virus. Another preferred Replikin forinclusion in an influenza vaccine is a Replikin demonstrated to havere-emerged in a strain of influenza after an absence of one or moreyears.

EXAMPLE 4

Analysis of sequence data of isolates of Plasmodium falciparum antigensfor the presence and concentration of Replikins is carried out by visualscanning of sequences or through use of a computer program based on the3-point recognition method described herein. Isolates of Plasmodiumfalciparum are obtained and the amino acid sequence of the protein isobtained by any art known method, such as by sequencing the gene andderiving the protein sequence therefrom. Sequences are scanned for thepresence of Replikins, conservation of Replikins over time andconcentration of Replikins in each isolate. This information providesdata that are used to form the basis for anti-malarial peptide vaccinesor nucleic acid based vaccines.

Peptide vaccines or nucleic acid-based vaccines based on the Replikinsobserved in the malaria causing organism are generated. Preferably, thepeptide or nucleic acid vaccine is based on or includes any Replikinsequences that are observed to be present on a surface antigen of theorganism. The vaccines may include any combination of Replikin sequencesidentified in the malaria causing strain.

For vaccine production, the Replikin peptide or peptides identified asuseful for an effective vaccine are synthesized by any method, includingchemical synthesis and molecular biology techniques, including cloning,expression in a host cell and purification therefrom. The peptides arepreferably admixed with a pharmaceutically acceptable carrier in anamount determined to induce a therapeutic antibody reaction thereto.Generally, the dosage is about 0.1 μg to about 10 mg.

Then malaria vaccine is preferably administered to a patient in needthereof at any time during the year, and particularly prior to travel toa tropical environment.

Another embodiment includes an antisense nucleic acid moleculecomplementary to the coding strand of the gene or the mRNA encodingorganism for the replikins in organisms including, but not limited to,viruses, trypanosomes, bacteria, fungi, algae, amoeba, and plants,wherein said antisense nucleic acid molecules is complementary to anucleotide sequence of a replikin containing organism.

1-173. (canceled)
 174. A peptide consisting of 23 to 50 amino acidresidues comprising any one of the sequences disclosed in the variableamino acid sequence of SEQ ID NO:
 698. 175. The peptide of claim 174wherein said peptide is an HIV peptide.
 176. The peptide of claim 175wherein said HIV peptide is a trans-activator peptide.
 177. The peptideof claim 174 consisting of 23 amino acid residues.
 178. A peptideconsisting of 19 to 50 amino acid residues comprising any one of thesequences beginning at position 5 and ending at position 23 of thevariable amino acid sequence of SEQ ID NO:
 698. 179. The peptide ofclaim 178 wherein said peptide is an HIV peptide.
 180. The peptide ofclaim 179 wherein said HIV peptide is a trans-activator peptide. 181.The peptide of claim 178 consisting of 19 amino acid residues.